Non-mevalonate isoprenoid pathway

ABSTRACT

The present invention is directed to enzymes and intermediates of the non-mevalonate isoprenoid pathway downstream of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream of isopentenyl pyrophosphate or dimethylallyl pyrophosphate. These are used as a basis for a screening method for inhibitors of these enzymes, and a method for identifying inhibitor-resistant variants thereof. Further disclosures refer to DNA coding for said enzymes and for inhibitor-resistant variants thereof, vectors containing said DNA, cells containing said vector, and plant seeds comprising cells containing said vector. This invention is useful for the inhibition of the biosynthesis of isoprenoids in plants, bacteria and protozoa, for conferring herbicide-resistance to plants, as well as for weed control in agriculture using a crop containing a herbicide-resistant gene and an effective amount of a suitable herbicide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/296,416, having a filing date of Jul. 8, 2003, which is a 35U.S.C. § 371 national phase application of International PCT ApplicationSerial No. PCT/EP01/06255 filed Jun. 1, 2001, which claims priority toGerman Patent Application No. 100 27 821.3, filed Jun. 5, 2000, thecontents of each of which are incorporated by reference as if recited infull herein.

FIELD OF THE INVENTION

The present invention relates to the non-mevalonate isoprenoid pathway.More particularly, it relates to intermediates downstream from2C-methyl-D-erythritol 2,4-cyclopyrophosphate and their production andto genes and proteins operative in their biosynthesis; as well aspurified isolated DNA coding for said proteins and expression vectorscontaining a sequence of such DNA as well as recombinant cellscontaining such vectors. Moreover, the present invention relates to ascreening method for detecting inhibitors of enzymatic conversionsdownstream from 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and toinhibitors, notably herbicides, detectable thereby as well ascompositions and processes for inhibiting the synthesis of isoprenoidsand for controlling the growth of organisms, notably plants based onsaid inhibitors. The invention also relates to the development ofinhibitor-resistant plant enzymes and plants, plant tissues, plant seedsand plant cells.

By the classical research of Bloch, Cornforth, Lynen and coworkers,isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)have been established as key intermediates in the biosynthesis ofisoprenoids via mevalonate. It has recently been detected that in mostbacteria, in protists like Plasmodium falciparum and in the plastids ofplants an alternative non-mevalonate pathway is operative. It has so faronly been partially explored. It can be conceptualized to consist ofthree segments:

In a first pathway segment shown in FIG. 1 pyruvate (1) andD-glyceraldehyde 3-phosphate (2) undergo a condensation to1-deoxy-D-xylulose 5-phosphate (DXP) (3) by 1-deoxy-D-xylulose5-phosphate synthase. Subsequently, DXP is converted to2C-methyl-D-erythritol 4-phosphate (4) by a two-step reaction comprisinga rearrangement and a reduction by 1-deoxy-D-xylulose 5-phosphatereductoisomerase. This establishes the 5-carbon isoprenoid skeleton.

In the subsequent segment of the non-mevalonate pathway (FIGS. 2 and 3),2C-methyl-D-erythritol 4-phosphate (4) is first condensed with CTP to4-diphosphocytidyl-2C-methyl-D-erythritol (5) in the presence ofmagnesium ions. This intermediate (5) is subsequently condensed with ATPto 4-diphosphocytidyl-2C-methyl-erythritol 2-phosphate (6) by4-diphosphocytidyl-2C-methyl-D-erythritol kinase in the presence ofmagnesium ions. The intermediate (6) is subsequently converted into2C-methyl-D-erythritol 2,4-cyclopyrophosphate (7). These three enzymaticsteps form a biosynthetic unit which activates the isoprenoidC₅-skeleton for the third pathway segment (Rodich et al. Proc. Natl.Acad. Sci. USA 96, 11758-11763 (1999); Lyttgen et al. Proc. Natl. Acad.Sci. USA 97, 1062-1067 (2000); Herz et al. Proc. Natl. Acad. Sci. USA97, 2486-2490 (2000)).

The third pathway segment is the subject of the present invention. Itconcerns the reductive conversion of 2C-methyl-D-erythritol2,4-cyclopyrophosphate into intermediates of the type IPP or DMAPP. Withthis segment, the non-mevalonate isoprenoid pathway (a trunk pathway) iscompleted. IPP and DMAPP subsequently undergo condensation into higherisoprenoids (or terpenoids).

The non-mevalonate isoprenoid pathway is wholly absent in animals. Thismakes it an ideal target for pesticidal or medical purposes. Theidiosyncratic nature of the reactions in this pathway reduces the riskof cross-inhibition with other, notably mammalian enzymes.

SUMMARY OF THE INVENTION

Therefore, it is a first object of the invention to provide a chemicalcompound which serves as intermediate in the non-mevalonate isoprenoidpathway downstream from 2C-methyl-D-erythritol 2,4-cyclopyrophosphateand upstream from the intermediates of the type IPP or DMAPP. Thisobject has been achieved by the compounds of claims 1 to 5 or by thecell fluid of claims 6 or 7.

It is a second object of the invention to provide a process forconverting 2C-methyl-D-erythritol 2,4-cyclopyrophosphate into anintermediate downstream from 2C-methyl-D-erythritol2,4-cyclopyrophosphate and upstream from the intermediates of the typeIPP or DMAPP. This object is achieved by the process according to claims8 to 10.

It is a third object of the invention to provide a method of screeningfor inhibitors of an enzyme operative downstream from2C-methyl-D-erythritol 2,4-cyclopyrophosphate. The object is achieved bythe methods of claims 17 to 19.

It is a fourth object of the invention to provide an enzyme in a formthat is functional in the non-mevalonate isoprenoid pathway downstreamfrom 2C-methyl-D-erythritol 2,4-cyclopyrophosphate. This object isachieved by the enzyme according to claim 12 or by a protein comprisingsuch enzyme.

It is a further object of the invention to provide a purified, isolatednucleic acid, notably DNA, coding for an enzyme in accordance with claim12 or a vector or recombinant cell derived therefrom. This object hasbeen achieved by the subject matter of claims 13 to 16.

It is a further object of the invention to provide a method foridentifying an inhibitor-resistant variant of one of the above enzymesas well as of nucleic acids and DNA vectors encoding said variants aswell as cells and seeds of plants harboring such vector as well as amethod for conferring inhibitor-resistance to plants and a correspondingmethod of weed control. These objects are achieved in accordance withclaims 20 to 28.

It is a further object of the invention to provide a novel inhibitor foran above-identified enzyme, compositions containing such inhibitor andmethods of in vivo inhibiting the biosynthesis of isoprenoids. Theseobjects are achieved in accordance with claims 29 to 31.

Wherever a phosphorylated compound or carboxylic acid compound ismentioned herein it may exist as a free acid or as a salt with at leastone proton replaced by ammonium or a metal ion or an organic cation. Themetal ion may preferably be an alkali metal ion or an alkaline earthmetal ion. The organic cation may be derived from an amine. It may be asulphonium ion, a guanidinium ion or a heteroaromatic ion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the previously established first pathway segment of thenon-mevalonate pathway.

FIGS. 2 and 3 show the previously established second pathway segment ofthe non-mevalonate pathway.

DETAILED DESCRIPTION OF THE INVENTION Synthesis of2C-Methyl-D-erythritol 2,4-Cyclopyrophosphate

2C-methyl-D-erythritol 2,4-cyclopyrophosphate is a key substance for theinvention. It may be obtained in the required amounts by a large-scaleenzymatic preparation. The immediate starting materials for thispreparation are pyruvate and D-glyceraldehyde 3-phosphate. However, itis preferred to use dihydroxyacetone phosphate in conjunction withtriosephosphate isomerase under appropriate conditions as a source forD-glyceraldehyde 3-phosphate. Still more advantageous is the use ofglucose and ATP in conjunction with the glycolytic enzymes hexokinase,phosphoglucose isomerase, phosphofructokinase, aldolase andtriosephosphate isomerase.

For the special case of dihydroxyacetone phosphate, the production of2C-methyl-D-erythritol 2,4-cyclopyrophosphate may be characterized asfollows:

-   -   (a) reacting dihydroxyacetone phosphate and sodium pyruvate in        the presence of magnesium salt, thiamine pyrophosphate, triose        phosphate isomerase and 1-deoxy-D-xylulose 5-phosphate synthase        to produce 1-deoxy-D-xylulose 5-phosphate;    -   (b) reacting the reaction mixture obtained in step (a) with        glucose and NADP⁺ in the presence of an Mg²⁺ or Mn²⁺ salt,        glucose dehydrogenase and 1-deoxy-D-xylulose 5-phosphate        reductoisomerase to produce 2C-methyl-D-erythritol 4-phosphate;    -   (c) reacting the reaction mixture obtained in step (b) with        cytidyl triphosphate, an enzyme as defined below and a divalent        metal salt to produce 4-diphosphocytidyl-2C-methyl-D-erythritol;    -   (d) reacting the reaction mixture obtained in step (c) with        adenosine triphosphate, a divalent metal salt and an enzyme as        defined below to produce        4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate;    -   (e) reacting the reaction mixture obtained in step (d) with an        enzyme as defined below to produce 2C-methyl-D-erythritol        2,4-cyclopyrophosphate;    -   (f) isolating the product of step (e).

For step (a) the enzyme 1-deoxy-D-xylulose 5-phosphate synthase isproduced in accordance with a previously described method from Bacillussubtilis (Williams et al., J. Bacteriol. 146 (3), 1162-1165 (1981)) andas exemplified herein.

Dihydroxyacetone phosphate is produced in accordance with Effenberger etal. (Tetrahedron Lett. 28, 1641-1644 (1987)). For step (b)1-deoxy-D-xylulose 5-phosphate reductoisomerase is produced inaccordance with a previously described method from E. coli. For step (c)the enzyme 4-diphosphocytidyl-2C-methyl-D-erythritol synthase isrequired. This enzyme is obtained by expressing the gene ygbP of E. colias described herein. For step (d) the enzyme4-diphosphocytidyl-2C-methyl-D-erythritol kinase is required. Thisenzyme is obtained by expressing the gene ychB of E. coli as describedherein.

For step (e) the enzyme 2C-methyl-D-erythritol 2,4-cyclopyrophosphatesynthase is required. It is obtained by expressing the gene ygbB of E.coli as described herein.

Steps (a) to (e) may be carried out as separate steps, optionally withintermediate isolation or as a one-pot reaction.

This synthesis may be carried out with any desired labelling, notablywith deuterium, tritium, 13-carbon, 14-carbon or 32-phosphorus.Preferred for the purposes of the present invention is a total orpartial labelling of 2C-methyl-D-erythritol 2,4-cycloprophosphate with13-carbon for the purpose of analytical discrimination or a labellingwith 14-carbon or 32-phosphorus for the purpose of detection, wherebythe 13-carbon labelling and the radioactive labelling may be combined.

Total ¹³C-labelling can be carried out advantageously starting from[U—¹³C₆] glucose and [U—¹³C₃] sodium pyruvate or [2,3-¹³C₂]pyruvate. Inthe presence of thiamine pyrophosphate, ATP and MgCl₂ the followingenzymes are used for preparing [U—¹³C₅]1-deoxy-D-xylulose 5-phosphate:triose phosphate isomerase, hexokinase, phosphoglucose isomerase,phosphofructokinase, aldolase and 1-deoxy-D-xylulose 5-phosphatesynthase. Subsequently, the product can be converted to[U—¹³C₅]2C-methyl-D-erythritol 4-phosphate with 1-deoxy-D-xylulose5-phosphate reductoisomerase, glucose dehydrogenase and glucose, NADP⁺and MgCl₂.

Further [U—¹³C₅]2C-methyl-D-erythritol 4-phosphate can be converted into[U—¹³C₅]4-diphosphocydidyl-2C-methyl-D-erythritol,[U—¹³C₅]4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and[U—¹³C₅]2C-methyl-D-erythritol 2,4-cyclopyrophosphate using the enzymesYgbP, YchB and YgbB in the presence of CTP, ATP, MgCl₂ and MnCl₂. Forregeneration of ATP it is possible to use also pyruvate kinase in thepresence of phosphoenol pyruvate.

Downstream Intermediates

It has been surprisingly found that an intermediate of thenon-mevalonate pathway, downstream from 2C-methyl-D-erythritol2,4-cyclopyrophosphate can be produced by using the latter compound asstarting material and cells or portions of cells as catalyst.

The cells used for producing the catalyst may be cells of any organismendowed with the non-mevalonate pathway, notably cells of plants,protists, like Plasmodium falciparum or bacteria. It is possible to useplastids instead of plant cells. A plant cell culture, for example ofCatharanthus roseus may be used.

In one preferred embodiment plastids of plants are used. The plastidsmay be chromoplasts, chloroplasts, etioplasts or leucoplasts.

The plastids may be obtained from plant tissue by disruption of thecells, filtration and centrifugation according to known methods by e.g.Camara (Methods Enzymol. 214, 352-365 (1993)) or Liedvogel (Cytobiology12, 155-174 (1976)). These publications also disclose incubation mediafor these plastids.

The incubation may be carried out in any incubation medium suitable forincubating plastid or cell suspensions. It is merely required that themetabolism is operative at least to the extent of the alternativebiosynthetic pathway. The incubation medium should support themetabolism. In the simplest and most preferred case the incubationmedium should not inhibit the reactions involved and all cofactors wouldbe provided by the plastids or cells. It is also possible to add one orseveral cofactors to the incubation medium. The pH of the incubationmedium should be physiologically suitable, preferably in the range of 5to 9 and especially in the range of 7 to 8.

Since the plastids are biochemically fully competent they will provideany additional components for the biosynthesis. It is preferred but notmandatory to add a magnesium salt and/or a divalent manganese salt forbringing the enzymes to optimum activity and it is further preferred toadd NADPH and/or NADP each in concentrations of 0.5 to 4 mM preferably 1to 2 mM. Further FAD may be added in a concentration of preferably 1 to100 μM, especially 5 to 50 μM. It is advantageous to add NaF in order toblock phosphatases. The concentration of NaF is preferably 1 to 20 mM.

The plastids or cells isolated by methods known per se may be completelyuninjured or more or less injured, even to the extent of being at leastpartially disrupted. The biochemical competence and the success of thescreening procedure is largely independent of such differences and quiterobust.

It has been surprisingly found that 2C-methyl-D-erythritol2,4-cyclopyrophosphate is taken up by plastids.

In another preferred embodiment plant cells or preferably plastidsthereof; or bacterial cells such as E. coli, notably strain M15 (pREP4),are grown and harvested by centrifugation; and subsequently resuspendedfor disruption. Any conventional disruption method may be used. Apreferred disruption method makes use of an incubation with lysozyme inan appropriate medium. The medium may for example comprise trishydrochloride, pH 7.5 to 8.5, preferably pH 8.0 as well as MgCl₂ (e.g. 5to 20 mM) and dithiothreitol (e.g. 2 to 10 mM).

Subsequently, the reaction mixture is cooled and sonified. The obtainedmixture is centrifuged to produce a pellet of solid components. It wassurprisingly found that the enzymatic activity for the conversion of2C-methyl-D-erythritol 2,4-cyclopyrophosphate is located in the solidcell fraction. Therefore, this solid cell fraction is resuspended in anappropriate medium (preferably the same as above).

It is possible to separate the solid cell fraction further insubfractions. This may be accomplished by conventional methods, forexample, by differential centrifugation or centrifugation with densitygradients.

Subsequently, 2C-methyl-D-erythritol 2,4-cyclopyrophosphate is added tothis suspension. For detection purposes it is preferred to includeradioactively labelled 2C-methyl-D-erythritol 2,4-cyclopyrophosphate. Itmay by T-, ¹⁴C- or ³²P-labelled. For example, it may be labelled with¹⁴C in position 2.

It is also possible to use a totally ¹³C-labelled starting material forfacilitating structural characterization of the product. To this end asmall proportion of radioactive (e.g. ¹⁴C-labelled) starting materialmay be added.

The mixture is incubated, preferably at 30 to 45° C. and especially at37° C. during a predetermined period of time, e.g. 2 to 20 h, especially4 to 12 h, more preferably 5 to 8 h.

It is preferable to add a cobalt-II salt, notably CoCl₂. Theconcentration of the cobalt-II salt should be preferably 1 to 30 mM andmore preferably 2 to 10 mM and especially about 5 mM. It has beensurprisingly found that the cobalt-II salt leads to an increasedconcentration of produced intermediate. The mechanism is presently notclear. It may be contemplated that the cobalt ions serve as promoter foran enzyme for the synthesis of the intermediate or as an inhibitor for asubsequent enzyme.

After cultivation, the mixture is centrifuged (optionally afterdisruption of the cells or plastids) and the aqueous supernatant isanalyzed. For this purpose, it may be concentrated e.g. bylyophilization. Aliquots of the concentrate may be analyzed bychromatography, notably high performance liquid chromatography (HPLC).It is preferred to employ ion pair, reversed phase HPLC. As a column, itis possible to use a Multospher 120 RP 18 column (5 μm crystallinesilica gel, 4.6×250 mm, CS-Chromatographie Service GmbH, Langerwehe,Germany). For establishing ion pair conditions the column should beequilibrated with an aqueous solution of a tetra-alkylammonium salt,preferably tetra-n-butylammonium salt, whereby a conventional anion,e.g. chloride or hydrogen sulfate may be used. The column developmentmay be effected with an aqueous solution (e.g. 5 to 20, preferably 10mM) of the above tetra-alkylammonium salt, whereby a (preferably linear)gradient of a water-soluble solvent, e.g. methanol may be used, e.g.from 0 to 42% (v/v) of methanol. The effluent is monitored with anappropriate detector, notably a radiodetector in the case ofradiolabelled starting material.

It has been observed that 2C-methyl-D-erythritol 2,4-cyclopyrophosphateis converted under the above conditions into a new intermediate which isdistinct from the starting material and also from IPP and DMAPP. It hasa retention volume between the retention volumes of2C-methyl-D-erythritol 2,4-cyclopyrophosphate and IPP. Moreover, whenthe reaction is followed over time the lowering of the peak of2C-methyl-D-erythritol 2,4-cyclopyrophosphate is clearly correlated withan increase of the product peak.

Enzymes of the Isoprenoid Pathway

We have previously demonstrated that the E. coli enzymes YgbP, YchB andYgbB and homologous enzymes in other organisms are in charge of theconversion of 2C-methyl-D-erythritol 4-phosphate to2C-methyl-D-erythritol 2,4-cyclopyrophosphate via4-diphosphocytidyl-2C-methyl-D-erythritol anddiphosphocytidyl-2C-methyl-D-erythritol 2-phosphate.

We have now used a set theoretical approach to determine a set ofenzymes in which with very high likelihood the enzymes operatingdownstream from 2C-methyl-D-erythritol 2,4-cyclopyrophosphate andupstream of IPP and/or DMAPP are situated. This has been accomplished bydetermining the genes which have the same taxonomic distribution asygbP, ychB and ygbB. Specifically, a first subset of genes(intersection) is determined which are common to the sequenced genomesof all “positive” organisms which are known to be endowed with thenon-mevalonate pathway. ygbP, ychB and ygbB were found to be members ofsaid first subset. Next, “negative” organisms like Saccharomycescerevisiae are selected which are known to lack the non-mevalonatepathway. Thereafter, a second subset of genes is determined bysubtracting from the first subset those genes which have a homologous inthe negative organisms. Thereafter a third subset was formed bysubtracting from the second subset any genes coding for proteins withwell established known function. The remaining narrowest subset consistsof the E. coli genes gcpE and lytB as designated in the E. coli genome.The taxonomic distribution of these two genes is shown in Tables 1 and2.

A slightly broader set of four enzymes includes additionally the E. coligenes yjeE and ybeB. The taxonomic distribution of these two genes isshown in Tables 3 and 4.

TABLE 1 Occurrence of orthologous gcpE sequences in various organismsCorresponding to gcpE^(a) Organism Accession number, basepairs Aquifexaeolicus VF5 gb^(b) AE000745, 352-1425 Bacillus subtilis 168 emb^(c)Z99116, 193139-194272 Chlamydia pneumoniae CWL029 gb AE001621, 8026-9867Chlamydia trachomatis D/UW-3/CX gb AE001280, 4785-6593 Deinococcusradiodurans gb AE001898, 7744-9033 Escherichia coli K-12 MG1655 gbAE000338, 372-1204 Haemophilus influenzae Rd gb U32712, 1613-2719Helicobacter pylori strain 26695 gb AE000577, 90-1169 Helicobacterpylori strain J99 gb AE001490, 107-1186 Mycobacterium leprae gb L78824,16209-17186 Mycobacterium tuberculosis H37Rv emb AL008883, 10845-12008Streptomyces coelicor A3(2) emb AL049485, 15496-16650 Synechocystis sp.PCC6803 dbj^(d) D90908, 73164-74375 Thermotoga maritima gb AE001754,6801-8306 Treponema pallidum gb AE001221, 10202-11416^(a)http://www.ncbi.nlm.nih.gov ^(b)GenBank database ^(c)europeandatabase ^(d)database of Japan

TABLE 2 Occurrence of orthologous lytB sequences in various organismsCorresponding to lytB^(a) Organism Accession number, basepairsAcinetobacter sp. BD413 gb^(b) AF027189, 264-1214 Aquifex aeolicus VF5gb AE000754, 71-940 Arabidopsis thaliana chromosome II BAC emb^(c)AL035521, 66098-68216 Bacillus subtilis 168 dbj^(d) D84432,122126-123070 Burkholderia pseudomallei gb AF098521, 236-1177Campylobacter jejuni NCTC 11168 emb X89371, 1655-2488 Chlamydiapneumoniae CWL029 gb AE001682, 5359-6291 Chlamydia trachomatis D/UW-3/CXgb AE001359, 2001-2924 Deinococcus radiodurans gb AE002049, 12853-13860Escherichia coli K-12 MG1655 gb AE000113, 5618-6565 Haemophilusinfluenzae Rd gb U32781, 8285-9229 Helicobacter pylori strain J99 gbAE001527, 7015-7839 Helicobacter pylori strain 26695 gb AE000556, 91-915Listeria monocytogenes gb U17284, 2082<>2555 Mycobacterium leprae embAL049491, 6002-7009 Mycobacterium tuberculosis H37Rv emb AL021897,63983-64990 Nicotiana tabacum gb AF159699, 1<>504 Pseudomonas aeruginosagb L76605, 6331<>6729 Pseudomonas fluorescens gb M35366, 1857>2396Synechocystis sp. PCC6803 dbj D64000, 46364-47584 Thermotoga maritima gbAE001796, 3613-4440 Treponema pallidum gb AE001230, 1785-2915^(a)http://www.ncbi.nlm.nih.gov ^(b)GenBank database ^(c)europeandatabase ^(d)database of Japan

TABLE 3 Occurrence of orthologous yjeE sequences in various organismsCorresponding to yjeE^(a) Organism Accession number, basepairs Anabaenaspecies PCC7120 gb^(b) AF046871, 990-1478 Aquifex aeolicus VF5 gbAE000710, 2932-3333 Bacillus subtilis 168 Dbj^(d) D88802, 27672-28148Borrelia burgdorferi gb AE001129, 5010-5423 Bradyrhizobium japonicum gbAF042096, 7016-7489 Campylobacter jejuni NCTC 11168 emb^(c) AL139076,361-768 Chlamydia pneumoniae CWL029 gb AE001648, 7612-8037 Chlamydiatrachomatis D/UW-3/CX gb AE001324, 7590-8063 Deinococcus radiodurans gbAE002066, 2143-2589 Escherichia coli K-12 MG1655 gb AE000489, 3299-3760Erysipelothrix rhusiopathiae dbj AB019247, 4372-4692 Haemophilusinfluenzae Rd gb U32692, 68-544 Helicobacter pylori strain J99 gbAE001497, 9098-9499 Helicobacter pylori strain 26695 gb AE000584,8919-9320 Lactococcus lactis emb Z70730, 4572-4916 Mycobacterium lepraegb U00020, 6462-6947 Mycobacterium tuberculosis H37Rv emb Z77165,27337-27843 Neisseria meningitidis strain Z2491 gb AF058689, 10961-11371Neisseria meningitidis strain Z4400 gb AF194079, 1685-2095 Rickettsiaprowazekii emb AJ235270, 11821-12255 Streptomyces coelicor emb AL031317,21836-22282 Synechocystis sp. PCC6803 dbj D90914, 142000-142473Thermotoga maritima gb AE001806, 7502-7987 Treponema pallidum gbAE001257, 7721-8128 ^(a)http://www.ncbi.nlm.nih.gov ^(b)GenBank database^(c)european database ^(d)database of Japan

TABLE 4 Occurrence of orthologous ybeB sequences in various organismsCorresponding to ybeB^(a) Organism Accession no., basepairs Aquifexaeolicus VF5 gb^(b) AE000732, 4157-4486 Bacillus subtilis 168 emb^(c)Z99117, 42657-43013 Borrelia burgdorferi gb AE001177, 8901-9260Campylobacter jejuni NCTC 11168 emb AL139078, 116712-117038 Chlamydiamuridarum gb AE002282, 5712-6071 Chlamydia pneumoniae CWL029 gbAE001671, 6617-6976 Chlamydia trachomatis D/UW-3/CX gb AE001349,5715-6074 Deinococcus radiodurans gb AE002087, 4944-5294 Escherichiacoli K-12 MG1655 gb AE000168, 6454-6663 Haemophilus influenzae Rd gbU32688, 11870-12178 Helicobacter pylori strain J99 gb AE001554,2536-2877 Helicobacter pylori strain 26695 gb AE000642, 600-941Mycobacterium tuberculosis H37Rv emb Z81368, 40124-40504 Neisseriameningitidis strain MC58 gb AE002552, 3227-3613 Neisseria meningitidisstrain Z2491 emb AL162753, 46997-47383 Rickettsia prowazekii embAJ235273, 148106-148432 Streptomyces coelicor emb AL136518, 16927-17373Synechocystis sp. PCC6803 dbj^(d) D90908, 56510-56974 Thermotogamaritima gb AE001700, 2645-2977 Treponema pallidum gb AE001245,10516-10851 ^(a)http://www.ncbi.nlm.nih.gov ^(b)GenBank database^(c)european database ^(d)database of Japan

With the functional assignments of YgbP, YchB and YgbB with theproduction of proteins having enzymatically competent foldingstructures, we have provided avenues for the production of the productsof the enzymatic reaction of YgbP, YchB and YgbB, namely4-diphosphocytidyl-2C-methyl-D-erythritol,4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and2C-methyl-D-erythritol 2,4-cyclopyrophosphate or salts thereof. Based onthis achievement, we have opened new avenues for the inhibition of thealternative isoprenoid pathway in plants as well as bacteria and also inprotozoa, like Plasmodium.

The designations of YgbP, YgbB and YchB in the E. coli genome are usedherein also for the homologous proteins in other organisms.

We have found that in plants, notably Arabidopsis thaliana the enzymeshomologous to YgbP, YchB and YgbB have a leader peptide not present inthe bacterial enzymes. This leader peptide serves the purpose oftransport of the enzyme into the plastids. Such specific leader sequencemay be replaced by any other leader sequence from A. thaliana or fromany other plant or it may also be eliminated.

The A. thaliana sequence of YgbP has been obtained by genome sequencing.We have sequenced the gene ygbP of A. thaliana and cloned thefull-length gene from RNA. The cDNA sequence of the cloned ygbP genefrom A. thaliana was different from the DNA sequence found in thedatabase (gb AC004136) due to introns. The amino acid sequencecorresponding to this cDNA is also different from the amino acidsequence given in the database (gb AC004136). This seems to be due toerroneous computational intron splicing from chromosomal DNA. The cDNAleader sequence was found to be identical to the database prediction.

The genes ygbP, ygbB and ychB of E. coli were obtained by PCR usingprimers with specific restriction sites. In this PCR reaction tworecognition sites for restriction enzymes are introduced at the 5′-endand at the 3′ end. The preferred recognition site is NcoI or EcoRI atthe 5′-end and PstI at the 3′ end. The amplified PCR fragment and anexpression vector are digested with the same restriction enzymes andligated together with T4-ligase to yield recombinant plasmid capable ofautonomous replication in the host microorganism. The recombinantplasmid is used to transform the host microorganism. The preferred hostis E. coli. The same method was used for the genes ygbP, ygbB and ychBof Arabidopsis thaliana and for ychB of tomato whereby the nucleotidesequence was modified for the codon usage of E. coli for highlyexpressed genes (without leader sequence).

Nucleic Acids, Vectors, Expression Systems and Polypeptides

In practicing the present invention, many techniques in molecularbiology, microbiology, recombinant DNA, and protein biochemistry such asthese explained fully in, for example, Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A practicalApproach, Volumes I and II, 1985 (Glover, ed.); OligonucleotideSynthesis, 1984, (Gait, ed.); Transcription and Translation, 1984 (Hamesand Higgins, eds.); A Practical Guide to Molecular Cloning; the seriesMethods in Enzymology (Academic Press, Inc.); and Protein Purification:Principles and Practice, Second Edition (Springer-Verlag, NY.) are used.

The present invention encompasses nucleic acid sequences encoding plantenzymes, enzymatically active fragments derived therefrom, and relatedderived sequences from other plant species. As used herein, a nucleicacid that is “derived from” a sequence refers to a nucleic acid sequencethat corresponds to a region of the sequence, sequences that arehomologous or complementary to the sequence, and “sequence-conservativevariants” and “function-conservative variants”.

Sequence-conservative variants are those in which a change of one ormore nucleotides in a given codon position results in no alteration inthe amino acid encoded at that position. Function-conservative variantsare those in which a given amino acid residue has been changed withoutaltering the overall conformation and function of the polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar physico-chemical properties (such as, for example,acidic, basic, hydrophobic, and the like). Enzyme fragments that retainenzymatic activity can be identified according to the methods describedherein, e.g, expression in E. coli followed by enzymatic assay of thecell extract.

Sequences derived from plants other than Arabidopsis thaliana can beisolated by routine experimentation using the methods and compositionsprovided herein. For example, hybridization of a nucleic acid comprisingall or part of Arabidopsis sequence under conditions of intermediatestringency (such as, for example, an aqueous solution of 2×SSC at 65°C.) to cDNA or genomic DNA derived from other plant species can be usedto identify homologues. cDNA libraries derived from different plantspecies are commercially available (Clontech, Palo Alto, Calif.;Stratagene, La Jolla, Calif.). Alternatively, PCR-based methods can beused to amplify related sequences from cDNA or genomic DNA derived fromother plants. Expression of the identified sequence in, e.g. E. coli,using methods described in more detail herein, is then performed toconfirm the enzymatic activity of the polypeptide encoded by thesequence. Accordingly, sequences derived from dicotyledonous andmonocotyledonous plants are within the scope of the invention.

The nucleic acids of the present invention include purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo-nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases. The nucleic acids maybe isolated directly from cells. Alternatively, PCR can be used toproduce the nucleic acids of the invention, using either chemicallysynthesized strands or genomic material as templates. Primers used forPCR can be synthesized using the sequence information provided hereinand can further be designed to introduce appropriate new restrictionsites, if desirable, to facilitate incorporation into a given vector forrecombinant expression.

The nucleic acids of the present invention may be flanked by naturalArabidopsis regulatory sequences, or may be associated with heterologoussequences, including promoters, enhancers, response elements, signalsequences, polyadenylation sequences, introns, 5′- and 3′-noncodingregions and the like. The nucleic acids may also be modified by manymeans known in the art. Non-limiting examples of such modificationsinclude methylation, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, and internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoromidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.). Nucleic acids may contain one or moreadditional covalently linked moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, poly-L-Lysine,etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g.,metals, radioactive metals, iron, oxidative metals, etc.), andalkylators. The nucleic acid may be derivatized by formation of a methylor ethyl phosphotriester or an alkyl phosphoramidate linkage.Furthermore, the nucleic acid sequences of the present invention mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising thedisclosed sequences or derivatives or fragments thereof. A large numberof vectors, including plasmid and fungal vectors, have been describedfor replication and/or expression in a variety of eukaryotic andprokaryotic hosts. Non-limiting examples include pKK plasmids(Clontech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.),or pRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriatehost cells, using methods disclosed or cited herein or otherwise knownto those skilled in the relevant art. Recombinant cloning vectors willoften include one or more replication systems for cloning or expression,one or more markers for selection in the host, e.g. antibioticresistance, and one or more expression cassettes. Suitable host cellsmay be transformed/transfected/infected as appropriate by any suitablemethod including electroporation, CaCl₂ mediated DNA uptake, tungaeinfection, microinjection, microprojectile, or other establishedmethods.

Appropriate host cells include bacteria, archaebacteria, fungi,especially yeast, plant and animal cells, especially mammalian cells. Ofparticular interest are E. coli, B. subtilis, Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, andimmortalized mammalian myeloid and lyphoid cell lines. Preferredreplication systems include M13, ColE1, SV40, baculovirus, lambda,adenovirus, and the like. A large number of transcription initiation andtermination regulatory regions have been isolated and shown to beeffective in the transcription and translation of heterologous proteinsin the various hosts. Examples of these regions, methods of isolation,manner of manipulation, etc. are known in the art. Under appropriateexpression conditions, host cells can be used as a source ofrecombinantly produced enzyme-derived peptides and polypeptides.

Advantageously, vectors may also include a transcription regulatoryelement (i.e. a promoter) operably linked to the enzyme portion. Thepromoter may optionally contain operator portions and/or ribosomebinding sites. Non-limiting examples of bacterial promoters compatiblewith E. coli include: trc promoter, β-lactamase (penicillinase)promoter; lactose promoter; tryptophan (trp) promoter; arabinose BADoperon promoter, lambda-derived P_(L) promoter and N gene ribosomebinding site; and the hybrid tac promoter derived from sequences of thetrp and lac UV5 promoters. Non-limiting examples of yeast promotersinclude 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GALI) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.Suitable promoters for mammalian cells include without limitation viralpromoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus(RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammaliancells may also require terminator sequences and poly A additionsequences, and enhancer sequences which increase expression may also beincluded. Sequences which cause amplification of the gene may also bedesirable. Furthermore, sequences that facilitate secretion of therecombinant product from cells, including, but not limited to, bacteria,yeast, and animal cells, such as secretory signal sequences and/orprohormone pro region sequences, may also be included.

Nucleic acids encoding wild-type or variant enzyme polypeptides may alsobe introduced into cells by recombination events. For example, such asequence can be introduced into a cell, and thereby effect homologousrecombination at the site of endogenous gene or a sequence withsubstantial identity to the gene. Other recombination-based methods,such as non-homologous recombinations or deletion of endogenous genes byhomologous recombination, may also be used.

Enzyme-derived polypeptides according to the present invention,including function-conservative enzyme variants may be isolated fromwild-type or mutant Arabidopsis cells, or from heterologous organisms orcells (including, but not limited to, bacteria, fungi, insect, plant,and mammalia cells) into which an enzyme-derived protein-coding sequencehas been introduced and expressed. Furthermore, the polypeptides may bepart of recombinant fusion proteins. Alternatively, polypeptides may bechemically synthesized by commercially available automated procedures,including, without limitation, exclusive solid phase synthesis, partialsolid phase methods, fragment condensation or classical solutionsynthesis.

“Purification” of an enzyme polypeptide refers to the isolation of theenzyme polypeptide in a form that allows its enzymatic activity to bemeasured without interference by other components of the cell in whichthe polypeptide is expressed. Methods for polypeptide purification arewell-known in the art, including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, reversed-phase HPLC, gelfiltration, ion exchange and partition chromatography, andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix. Alternatively, antibodies producedagainst the enzyme or against peptides derived therefrom can be used aspurification reagents. Other purification methods are possible.

The present invention also encompasses derivatives and homologues of theenzyme polypeptides. For some purposes, nucleic acid sequences encodingthe peptides may be altered by substitutions, additions, or deletionsthat provide for functionally equivalent molecules, i.e.,function-conservative variants. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofsimilar properties, such as, for example, positively charged amino acids(arginine, lysine, and histidine); negatively charged amino acids(aspartate and glutamate); polar neutral amino acids; and non-polaramino acids.

The designations of YgbP, YgbB and YchB in the E. coli genome are usedherein also for the homologous proteins in other organisms.

The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

Genes corresponding to YgbP (or YgbB) or YchB from any plant may bereadily isolated by well known techniques, for example by Southernhybridization or by PCR using degenerated primers. Notably, a cDNAlibrary of this plant in question is screened using the nucleic aciddirect labelling and detection system kit supplied byAmersham-Pharmacia-Biotech (Heidelberg, Germany). Hybridizationconditions are for example 7% sodium dodecyl sulfate (SDS). Positivelyhybridizing plaques are detected by luminescence detection (or in othersystems by autoradiography). After purification to single plaques, cDNAinserts are isolated, and their sequences determined by the chaintermination method using dideoxy terminators labeled with fluorescentdyes (Applied Biosystems, Inc., Foster City, Calif.). This experimentalprotocol can be used by one of ordinary skill in the art to obtain genessubstantially similar to the Arabidopsis gene from any other plantspecies.

Screening Methods to Identify Enzyme Inhibitors/Herbicides

The methods and compositions of the present invention can be used toidentify compounds that inhibit the function of the enzymes and thus arefor example useful as herbicides or as lead compounds for thedevelopment of useful herbicides. This may be achieved by providing acell that expresses the enzyme and thereby produces cell culturesexpressing the enzyme which are incubated in the presence of testcompounds to form test cultures, and in the absence of test compounds toform control cultures. Incubation is allowed to proceed for a sufficienttime and under appropriate conditions to allow for interference withenzyme function. At a predetermined time after the start of incubationwith a test compound, an assay is performed to monitor enzymaticactivity. In one embodiment, enzyme activity is monitored in wholecells. Alternatively, enzymatic activity may be monitored in cellextracts or media containing the isolated enzyme using assays such asthat described below. Additional controls, with respect to both culturesamples and assay samples, are also included, such as, for example, ahost cell not expressing the enzyme (e.g., a host cell transformed withan expression plasmid containing the enzyme gene in a reverseorientation or with no insert). Enzyme inhibitory compounds areidentified as those that reduce enzyme activity in the test culturesrelative to the control cultures.

Host cells that may be used in practicing the present invention includewithout limitation bacterial, fungal, insect, mammalian, and plantcells. Preferably, bacterial cells are used. Most preferably, thebacterial cell is a variant (such as, e.g. the imp mutant of E. coli)that exhibits increased membrane permeability for test compoundsrelative to a wild-type host cell.

Preferably, the methods of the present invention are adapted to ahigh-throughput screening, allowing a multiplicity of compounds to betested in a single assay. Such inhibitory compounds may be found in, forexample, natural product libraries, fermentation libraries (encompassingplants and microorganisms), combinatorial libraries, compound files, andsynthetic compound libraries. For example, synthetic compound librariesare commercially available from Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich Chemical Company, Inc. (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant, and animal extracts are available from, forexample, Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or arereadily producible. Additionally, natural and synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical, and biochemical means (Blondell et al. TibTech 14,60 (1996)). Inhibitor assays according to the present invention areadvantageous in accommodating many different types of solvents and thusallowing the testing of compounds from many sources.

Once a compound has been identified by the methods of the presentinvention as inhibitor, in vivo and in vitro tests may be performed tofurther characterize the nature and mechanism of the inhibitoryactivity. The effect of an identified compound on in vitro enzymaticactivity of purified or partially purified protein may be determined andenzyme kinetic plots may be used to distinguish, e.g., competitive andnon-competitive inhibitors.

Compounds identified as inhibitors using the methods of the presentinvention may be modified to enhance potency, efficacy, uptake,stability, and suitability for use in commercial herbicide applications,etc. These modifications are achieved and tested using methodswell-known in the art.

Isolation of Herbicide-Resistant Enzyme Variants

The present invention encompasses the isolation of enzyme variants thatare resistant to the action of enzyme inhibitors/herbicides. The enzymevariants may be naturally occurring or may be obtained by random orsite-directed mutagenesis.

In one embodiment, a population of cells or organisms expressing theenzyme of interest is mutagenized using procedures well-known in theart, after which the cells or organisms are subjected to a screening orselection procedure to identify those that are resistant to the toxiceffects of an inhibitor. The variant enzyme gene is then isolated fromthe resistant cell or organism using, e.g., PCR techniques.

In another embodiment, an isolated enzyme gene is subjected to random orsite-directed mutagenesis in vitro, after which mutagenized versions ofthe gene are reintroduced into an appropriate cell such as, e.g., E.coli, and the cells are subjected to a selection or screening procedureas above.

The variant enzyme genes are expressed in an appropriate host cell, andthe enzymatic properties of variant enzyme polypeptides are compared tothe wild-type enzyme. Preferably, a given mutation results in an enzymevariant polypeptide that retains in vitro enzymatic activity, whileexhibiting catalytic activity that is relatively more resistant to theselected herbicide(s) than is wild-type enzyme. Preferably, whenexpressed in a cell that requires enzyme activity for viability, thevariant exhibits (i) catalytic activity alone sufficient to maintain theviability of a cell in which it is expressed; or catalytic activity incombination with any herbicide resistant enzyme variant protein alsoexpressed in the cell, which may be the same as or different than thefirst enzyme protein, sufficient to maintain the viability of a cell inwhich it is expressed; and (ii) catalytic activity that is moreresistant to the herbicide than is wild-type enzyme.

Therefore, any one specific enzyme variant protein need not have thetotal catalytic activity necessary to maintain the viability of thecell, but must have some catalytic activity in an amount, alone or incombination with the catalytic activity of additional copies of the sameenzyme variant and/or the catalytic activity of other enzyme variantprotein(s), sufficient to maintain the viability of a cell that requiresenzyme activity for viability. For example, catalytic activity may beincreased to minimum acceptable levels by introducing multiple copies ofa variant encoding gene into the cell or by introducing the gene whichfurther includes a relatively strong promoter to enhance the productionof the variant.

More resistant means that the catalytic activity of the variant isdiminished by the herbicide(s), if at all, to a lesser degree thanwild-type enzyme catalytic activity is diminished by the herbicide(s).Preferred more resistant variant enzyme retains sufficient catalyticactivity to maintain the viability of a cell, plant, or organism whereinat the same concentration of the same herbicide(s), wild-type enzymewould not retain sufficient catalytic activity to maintain the viabilityof the cell, plant or organism.

Preferably, the catalytic activity in the absence of herbicide(s) is atleast about 5% and, most preferably, is more than about 20% of thecatalytic activity of the wild-type enzyme in the absence ofherbicide(s).

Herbicide-resistant enzyme variants can be used as genetic markers inany cell that is normally sensitive to the inhibitory effects of theherbicide formation. In one embodiment, DNA encoding anherbicide-resistant enzyme variant is incorporated into a plasmid underthe control of a suitable promoter. Any desired gene can then beincorporated into the plasmid, and the final recombinant plasmidintroduced into an herbicide-sensitive cell. Cells that have beentransformed with the plasmid are then selected or screened by incubationin the presence of a concentration of herbicide sufficient to inhibitgrowth and/or pigment formation.

Chemical-Resistant Plants and Plants Containing Variant Enzyme Genes

The present invention encompasses transgenic cells, including, but notlimited to seeds, organisms, and plants into which genes encodingherbicide-resistant enzyme variants have been introduced. Non-limitingexamples of suitable recipient plants are listed in Table 5 below:

TABLE 5 RECIPIENT PLANTS COMMON NAME FAMILY LATIN NAME Maize GramineaeZea mays Maize; Dent Gramineae Zea mays dentiformis Maize, FlintGramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microspermaMaize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea maysamyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, WaxyGramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum speltaWheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticumturgidum Wheat, Large Spelt Pooideae Triticum spelta Wheat, PolishPooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidumWheat, singlegrained Pooideae Triticum monococcum Wheat, Small SpeltPooideae Triticum monococcum Wheat, Soft Pooideae Triticum aestivum RiceGramineae Oryza sativa Rice, American Wild Gramineae Zizania aquaticaRice, Australian Gramineae Oryza australiensis Rice, Indian GramineaeZizania aquatica Rice, Red Gramineae Oryza glaberrima Rice, TuscaroraGramineae Zizana aquatica Rice, West African Gramineae Oryza glaberrimaBarley Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeumirregulare intermediate, also Irregular Barley, Ancestral TworowPooideae Hordeum spontaneum Barley, Beardless Pooideae Hordeumtrifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley,fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed PooideaeHordeum vulgare hexastichon Barley, Tworrowed Pooideae Hordeum distichonCotton, Abroma Dicotyledoneae Abroma augusta Cotton, American UplandMalvaceae Gossypium hirsutum Cotton, Asiatic Tree also MalvaceaeGossypium arboreum Indian Tree Cotton, Brazilian, also, MalvaceaeGossypium barbadense Kidney, and, Pernambuco brasiliense Cotton, LevantMalvaceae Gossypium herbaceum Cotton Long Silk, also Malvaceae Gossypiumbarbadense Long Staple, Sea Island Cotton Mexican, also MalvaveaeGossypium hirsutum Short Staple Soybean, Soya Leguminosae Glycine maxSugar beet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plantArenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, CherrySolanaceae Lycopersicon esculentum cerasiforme Tomato, Common SolanaceaeLycopersicon esculentum commune Tomato, Currant Solanaceae Lycopersiconpimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato,Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersiconesculentum pyriforme Tomato, Tree Solanaceae Cyphomandra betacea PotatoSolanaceae Solanum tuberosum Potato, Spanish, Sweet ConvolvulaceaeIpormoca batatas potato Rye, Common Pooideae Secale cereale Rye,Mountain Pooideae Secale montanum Pepper, Bell Solanaceae Capsicumannuum grossum Pepper, Bird, also Solanaceae Capsicum annuum Cayenne,Guinea minimum Pepper, Bonnet Solanaceae Capsicum sinense Pepper,Bullnose, also Solanaceae Capsicum annuum Sweet grossum Pepper, CherrySolanaceae Capiscum annuum cerasiforme Pepper, Cluster, also RedSolanaceae Capsicum annuum Cluster fasciculatum Pepper, Cone SolanaceaeCapsicum annuum conoides Pepper, Goat, also Spur Solanaceae Capsicumfrutescens Pepper, Long Solanaceae Capsicum frutescens longum Pepper,Ornamental Red, Solanaceae Capsicum annuum also Wrinkled abbreviatumPepper, Tabasco Red Solanaceae Capsicum annuum conoides Lettuce, GardenCompositae Lactuca sativa Lettuce, Asparagus, also Compositae Lactucasativa asparagina Celery Lettuce, Blue Compositae Lactuca perennisLettuce, Blue, also Chicory Compositae Lactuca pulchella Lettuce,Cabbage, also Compositae Lactuca satica capitata Head Lettuce, Cos, alsoCompositae Lactuca sativa longifolia Longleaf, Romain Lettuce, Crinkle,also Compositae Lactuca sativa crispa Curled, Cutting, Leaf CeleryUmbelliferae Apium graveolens dulce Celery, Blanching, also UmbelliferaeApium graveolens dulce Garden Celery, Root, also Umbelliferae Apiumgraveolens Turniproote rapaceum Eggplant, Garden Solanaceae Solanummelongena Sorghum Sorghum All crop specie Alfalfa Leguminosae Medicagosativum Carrot Umbelliferae Daucus carota sativa Bean, ClimbingLeguminosae Phaseolus vulgaris vulgaris Bean, Sprouts LeguminosaePhaseolus aureus Bean, Brazilian Broad Leguminosae Canavalia ensiformisBean, Broad Leguminosae Vicia faba Bean, Common, also LeguminosaePhaseolus vulgaris French, White, Kidney Bean, Egyptian LeguminosaeDolichos lablab Bean, Long, also Yardlong Leguminosae Vignasesquipedalis Bean, Winged Leguminosae Psophocarpus teragonolobus Oat,also Common, Side, Avena Sativa Tree Oat, Black, also Bristle, AvenaStrigosa Lopsided Oat, Bristle Avena Pea, also Garden, Green,Leguminosae Pisum, sativum sativum Shelling Pea, Blackeyed LeguminosaeVigna sinensis Pea, Edible Podded Leguminosae Pisum sativum axipluumPea, Grey Leguminosae Pisum sativum speciosum Pea, Winged LeguminosaeTetragonolobus purpureus Pea, Wrinkled Leguminosae Pisum sativummeduilare Sunflower Compositae Helianthus annuus Squash, Autumn, WinterDicotyledoneae Cucurbita maxima Squash, Bush, also DicotyledoneaeCucurbita pepo melopepo Summer Squash, Turban Dicotyledoneae Cucurbitamaxima turbaniformis Cucumber Dicotyledoneae Cucumis sativus Cucumber,African, also Momordica charantia Bitter Cucumber, Squirting, also WildEcbalium elaterium Cucumber, Wild Cucumis anguria Poplar, CaliforniaWoody-Plant Populus trichocarpa Poplar, European Black Populus nigraPoplar, Gray Populus canescens Poplar, Lombardy Populus italica Poplar,Silverleaf, also Populus alba White Poplar, Wester Balsam Populustrichocarpa Tobacco Solanaceae Nicotiana Arabidopsis Thaliana CruciferaeArabidopsis thaliana Turfgrass Lolium Turfgrass Agrostis Other familiesof turfgrass Clover Leguminosae

Expression of the variant polypeptides in transgenic plants confers ahigh level of resistance to herbicides allowing the use of theseherbicides during cultivation of the transgenic plants.

Methods for the introduction of foreign genes into plants are known inthe art. Non-limiting examples of such methods include Agrobacteriuminfection, particle bombardment, polyethylene glycol (PEG) treatment ofprotoplasts, electro-poration of protoplasts, microinjection,macroinjection, tiller injection, pollen tube pathway, dry seedinhibition, laser perforation, and electrophoresis. These methods aredescribed in, for example, Jenes et al., and Ritchie et al. In:Transgenic Plants, Vol. 1, Engineering and Utilization, (Kung and Wu,eds.), Academic Press, Inc., Harcourt Brace Jovanovich 1993; andMannonen et al., Critical Reviews in Biotechnology, 14, 287-310 (1994).

In a preferred embodiment, the DNA encoding a variant enzyme is clonedinto a DNA vector containing an antibiotic resistance marker gene, andthe recombinant enzyme DNA-containing plasmid is introduced intoAgrobacterium tumefaciens containing a Ti plasmid. This “binary vectorsystem” is described in, for example, U.S. Pat. No. 4,490,838 and in Anet al. (Plant Mol. Biol. Manual A3 1-19 (1988)). The transformedAgrobacterium is then co-cultivated with leaf disks from the recipientplant to allow infection and transformation of plant cells. Transformedplant cells are then cultivated in regeneration medium, which promotesthe formation of shoots, first in the presence of the appropriateantibiotic to select for transformed cells, then in the presence ofherbicide. In plant cells successfully transformed with DNA encodingherbicide-resistant enzyme, shoot formation occurs even in the presenceof levels of herbicide that inhibit shoot formation from non-transformedcells. After confirming the presence of variant enzyme DNA using, forexample, polymerase chain reaction (PCR) analysis, transformed plantsare tested for their ability to withstand herbicide spraying and fortheir capabilities for seed germination and root initiation andproliferation in the presence of herbicide.

The methods and compositions of the present invention can be used forthe production of herbicide-resistant enzyme variants, which can beincorporated into plants to confer selective herbicide resistance on theplants. Intermediate variants of enzyme (for example, variants thatexhibit sub-optimal specific activity but high herbicide resistance, orthe converse) are useful as templates for the design ofsecond-generation enzyme variants that retain adequate specific activityand high resistance.

Herbicide resistant enzyme genes can be transformed into crop species insingle or multiple copies to confer herbicide resistance. Geneticengineering of crop species with reduced sensitivity to herbicides can:

-   -   (1) Increase the spectrum and flexibility of application of        specific effective and environmentally benign herbicides;    -   (2) Enhance the commercial value of these herbicides;    -   (3) Reduce weed pressure in crop fields by effective use of        herbicides on herbicide resistant crop species and a        corresponding increase in harvest yields;    -   (4) Increase sales of seed for herbicide resistant plants;    -   (5) Increase resistance to crop damage from carry-over of        herbicides applied in previous planting;    -   (6) Decrease susceptibility to changes in herbicide        characteristics due to adverse climate conditions; and    -   (7) Increase tolerance to unevenly or mis-applied herbicides.        For example, plants containing transgenic enzyme variant protein        can be cultivated. The crop can be treated with a weed        controlling effective amount of the herbicide to which the        enzyme variant transgenic plant is resistant, resulting in weed        control in the crop without detrimentally affecting the        cultivated crop.

The compounds detected as inhibitors by the above screening methods maybe used as pure compounds or in combination together with appropriateadditives for inhibiting the enzymes in plant, bacterial or protozoalorganisms. Conventional additives in the field of herbicides,antibacterial agents or antiprotozoal agents may be used.

The invention shall now be described with reference to specificexamples.

Reference Example 1

2.0 μg of the vector pQE30 (Qiagen, Hilden, Germany) is digested with 30U of NcoI (New England Biolabs (NEB), Schwalbach, Germany) in a totalvolume of 60 μl containing 6 μl of NEB4 buffer. The reaction mix isincubated for 3 h at 37° C. After adding 33 μM of each dNTP (NEB) and 5U Klenow fragment of polymerase I from E. coli (NEB) the reaction mix isincubated for additional 30 min at 25° C. The vector DNA is purifiedusing the PCR purification kit from Qiagen. 500 μl of buffer PB (Qiagen)is added to 98 μl of PCR reaction mixture and applied to a Qiaquickcolumn and centrifuged for 1 min at 14,000 rpm. The flow through isdiscarded. 0.75 ml of buffer PE (Qiagen) is loaded on the column andcentrifuged as before. The flow through is discarded and the column iscentrifuged for an additional 1 min at 14,000 rpm. The column is placedin a clean 1.5 ml eppendorf tube. 50 μl of H₂O (redistilled, sterile) isadded to the column and it is centrifuged for 1 min at 14,000 rpm. Theflow through contained 1.5 μg of purified vector DNA.

20 ng of vector DNA is religated with 1 U of T4-Ligase from Gibco-BRL(Eggenstein, Germany), 2 μl, of T4-Ligase buffer (Gibco-BRL) in a totalvolume of 10 μl yielding the plasmid pQE_noNco. The ligation mixture isincubated overnight at 4° C. With 2 μl of the ligation mixtureelectrocompetent E. coli XLI-Blue (Bullock et al. XL1-Blue: a highefficiency plasmid transforming recA Escherichia coli withβ-galactosidase selection. BioTechniques 5, 376-379 (1987); commercialsource: Stratagene, LaJolla, Calif., USA) cells are transformed.

Preparation of electrocompetent cells: 1 liter of Luria Bertani (LB)medium is inoculated 1:100 with fresh overnight culture. The cells aregrown at 37° C. with shaking at 220 rpm to an optical density of 0.5 at600 nm. The cells are chilled on ice for 20 min and centrifuged for 15min at 4,000 rpm at 4° C. The supernatant is removed and the pellet isresuspended in 1 liter of ice-cold sterile 10% (v/v) glycerol. The cellsare centrifuged two times as described before resuspending the cells in0.5 liter and in 20 ml of ice-cold sterile 10% (v/v) glycerol,respectively. The cells are centrifuged an additional time and thepellet is resuspended in a volume of 2 ml of ice-cold 10% (v/v)glycerol. This suspension is frozen in aliquots of 80 μl and stored inliquid nitrogen.

Electro-Transformation Using the Gene Pulser Apparatus from Biorad(Munich, Germany):

The electrocompetent cells are thawed on ice. 40 μl of the cellsuspension are mixed with 2 μl of ligation mixture and transferred intoa prechilled, sterile 0.2 cm cuvette (Biorad). The suspension is shakedto the bottom and the cuvette is placed into the prechilled chamberslide. The chamber slide is pushed into the chamber and the cells arepulsed at 2.50 kV, 25 μF and Pulse Controller setting 200Ω. The cuvetteis removed from the chamber and the cells are suspended in 1 ml of SOCmedium (2% (w/v) casein hydrolysate, 0.5% (w/v) yeast extract, 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose. Thesuspension is shaked for 1 h at 37° C. and 100 μl of the suspension isplated on LB plates containing 150 mg/l ampicillin for maintenance ofthe plasmid pQE_noNco.

Cells of Escherichia coli XL1-Blue harboring the vector pQE_noNco, aregrown overnight in LB medium containing 180 mg/l of ampicillin formaintenance of the plasmid in the host cells. 7 ml of the culture arecentrifuged for 20 min at 5,000 rpm. The cell pellet is used forisolation of the plasmid pQE_noNco with the mini plasmid isolation kitfrom Qiagen (Hilden, Germany). The pellet is resuspended in 0.3 ml of 10mM EDTA in 50 mM tris hydrochloride, pH 8.0. 30 μg RNase A are added.0.3 ml of 1% (w/v) SDS in 200 mM sodium hydroxide are added andincubated for 5 min at room temperature. 0.3 ml of chilled 3.0 M sodiumacetate, pH 5.5 are added and incubated for 10 min on ice. The mixtureis centrifuged for 15 min at 14,000 rpm in a minifuge. The supernatantis applied onto a Qiagen-tip 20, which is previously equilibrated with 1ml of 750 mM NaCl, 15% (v/v) ethanol and 0.15% (v/v) Triton X-100 in 50mM MOPS, pH 7.0. The Qiagen-tip is washed four times with 1 ml of 1000mM NaCl and 15% (v/v) ethanol in 50 mM MOPS, pH 7.0. The DNA is elutedwith 0.8 ml of 1250 mM NaCl and 15% (v/v) ethanol in 50 mM trishydrochloride, pH 8.5. The DNA is precipitated with 0.56 ml ofisopropanol, centrifuged 30 min at 14,000 rpm and washed with 1 ml ofice-cold 70% (v/v) ethanol. After drying in a speedvac for 5 min, theDNA is dissolved in 50 μl of redistilled H₂O. The solution contained 8.3μg of the vector DNA pQE_noNco.

The DNA of the vector pQE_noNco is sequenced by the automatedideoxynucleotide method (Sanger et al. Proc. Natl. Acad. Sci. USA 74,5463-5468 (1977)) using an ABI Prism 377™ DNA sequencer from PerkinElmer (Norwalk, USA) with the ABI Prism™ Sequencing Analysis Softwarefrom Applied Biosystems Divisions (Foster City, USA).

The DNA sequence is found to be as expected.

2.0 μg of the vector pQE_noNco is digested with 30 U of EcoRI and 30 Uof SalI (NEB) in a total volume of 60 μl containing 6 μl of EcoRI buffer(NEB). The reaction mix is incubated for 3 h at 37° C. The vector DNA ispurified using the PCR purification kit from Qiagen.

25 pmol of the oligonucleotides 5′-CACACAGAATTCATTAAAGAG GAGAAATTAACCATGGGAGGATCCGTCGACCTGCAGCC-3′ and 5′-GGCTGCAGGTCGACGGATCCTCCCATGGTTAATTTCTCCTCTTTA ATGMTTCTGTGTG-3′ are dissolved in 6 μlEcoRI buffer (NEB) and 54 μl H₂O. The solution is heated at 96° C. for 2min and cooled down to 10° C. within 12 h in order to hybridize the DNAlinker. The reaction mix is supplied with 30 U of EcoRI and 30 U of SalI(NEB) and incubated for 3 h at 37° C. The reaction mix is heated to 65°C. for 30 min in order to inactivate the enzymes and cooled down to 10°C. within 12 h for hybridisation. The reaction mix containsapproximately 730 ng of the DNA linker.

20 ng of the digested pQE_noNco vector DNA (see above) and 300 pg of theDNA linker, 2 μl of T4-Ligase buffer (Gibco-BRL) are ligated togetherwith 1 U of T4-Ligase from Gibco-BRL (Eggenstein, Germany), 2 μl ofT4-Ligase buffer (Gibco-BRL) in a total volume of 10 μl yielding theplasmid pNCO113. The ligation mixture is incubated overnight at 4° C.With 2 μl of the ligation mixture electrocompetent E. coli XL1-Bluecells are transformed.

5 μg of the plasmid pNCO113 are isolated and the DNA sequence of thevector pNCO113 is sequenced as described above. The culture is ondeposit with ATCC as a patent deposit with the title Escherichia colistrain XL1-Blue habouring plasmid pNCO113, assigned PTA-852, date ofdeposit: Oct. 14, 1999.

Reference Example 2 Production of an Expression Clone and Constructionof an Expression Vector for 1-deoxy-D-xylulose 5-phosphate Synthase ofBacillus subtilis

The expression vector pNCO113 is isolated as described in referenceexample 1. Chromosomal DNA from Bacillus subtilis strain BR151 (Williamset al. J. Bacteriol. 146(3), 1162-1165 (1981)) is isolated according toa method described in reference example 8.

The putative ORF yqiE coding for 1-deoxy-D-xylulose 5-phosphate synthaseof B. subtilis (accession no. dbj D84432) from basepair (bp) position193991 to 195892 is amplified by PCR using chromosomal B. subtilis DNAas template. The reaction mixture contained 25 pmol of primerTGATCCGCCATGGATCTTTTATCAATACAGG, 25 pmol of primer TTGAATAGAGGATCCCCGCC,20 ng of chromosomal DNA, 2 U of Taq DNA polymerase (Eurogentec,Seraing, Belgium) and 20 nmol of dNTPs in a total volume of 1.5 mMMgCl₂, 50 mM KCl, 10 mM tris hydrochloride, pH 8.8 and 0.1% (w/w) TritonX-100 in a total volume of 100 μl.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 60sec at 94° C., 60 sec at 50° C. and 120 sec at 72° C. follow. Afterfurther incubation for 20 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis. The PCRamplificate is purified with the PCR purification kit from Qiagen. 500μl of buffer PB (Qiagen) are added to 98 μl of PCR reaction mixture andapplied to a Qiaquick column and centrifuged for 1 min at 14,000 rpm.The flow through is discarded. 0.75 ml of buffer PE (Qiagen) are loadedon the column and centrifuged as before. The flow through is discardedand the column is centrifuged for an additional 1 min at 14,000 rpm. Thecolumn is placed in a clean 1.5 ml eppendorf tube. 50 μl of H₂O(redistilled, sterile) are added to the column and it is centrifuged for1 min at 14,000 rpm. The flow through contained 1.8 μg of purified PCRproduct.

2.0 μg of the vector pNCO113 and 1.8 μg of the purified PCR product aredigested in order to produce DNA fragments with overlapping ends. Eachrestriction mixture contained 7 μl of SalI buffer from (NEB), 7 μg ofBSA (NEB), 40 U of NcoI (NEB), 30 U of SalI (NEB) in a total volume of70 μl and is incubated for 3 h at 37° C. Digested vector DNA and PCRproduct are purified using the PCR purification kit from Qiagen.

20 ng of vector DNA and 34 ng of PCR product are ligated together with 1U of T4-Ligase from Gibco-BRL (Eggenstein, Germany), 2 μl of T4-Ligasebuffer (Gibco-BRL) in a total volume of 10 μl yielding the plasmidpNCODXSBACSU. The ligation mixture is incubated overnight at 4° C. With2 μl of the ligation mixture electrocompetent E. coli XL1-Blue cells aretransformed as described in example 2. 6 μg of plasmid DNA pNCODXSBACSUwere isolated.

The DNA insert of the plasmid PNCODXSBACSU is sequenced as described inreference example 1. The sequence is identical with the sequence foundin the database (accession no. dbj D84432).

Reference Example 3 Preparation and Purification of Recombinant1-deoxy-D-xylulose 5-phosphate Synthase of B. subtilis.

E. coli XL1-Blue cells harboring the plasmid pNCODXSBACSU are grown,induced, harvested and stored as described in reference example 9.

2 g of the cells are thawed in 10 ml of 25 mM tris-HCl pH 8.0 containing1 mM dithioerythritol, 10 mM EDTA and 6 mM phenylmethylsulfonyl fluoridein the presence of 1 mg lysozyme. The mixture is incubated at 37° C. for0.5 h, cooled on ice and sonified 6×10 sec with a Branson Sonifier 250(Branson SONIC Power Company, Danbury, USA), control value of 4 output.The suspension is centrifuged at 15,000 rpm at 4° C. for 30 min. Thesupernatant is applied on a column of Sepharose QFF (26 cm³, AmershamPharmacia Biotech, Freiburg, Germany) previously equilibrated with 200ml 25 mM tris-HCl pH 8.0 containing 0.5 mM MgCl₂ and 0.03% sodium azide(buffer A). The column is washed with 60 ml buffer A monitoring theextinction at 280 nm. 1-deoxy-D-xylulose 5-phosphate synthase is elutedfrom the column with a gradient from 0-1 M sodium chloride in 300 ml ofbuffer A. The enzyme is identified by SDS-PAGE showing a band at 68 kDa.Fractions showing this protein band are collected and dialysed againstbuffer A overnight.

The enzyme is further purified on a column of hydroxyl apatite (Macropep 40 μm (size 2.5×6 cm, Biorad, Munich, Germany) equilibrated withbuffer A. The enzyme is eluted by a gradient of 0-1 M potassiumphosphate, pH 6.5. The homogeneity of 1-deoxy-D-xylulose 5-phosphatesynthase is judged by SDS-PAGE. A prominent band at 67 kDa is visible,which is in agreement to the calculated molecular mass. The yield ofpure 1-deoxy-D-xylulose 5-phosphate synthase is 44 mg.

Reference Example 4 Determination of 1-deoxy-D-xylulose 5-phosphateSynthase Activity 1. By Nuclear Magnetic Resonance (NMR)

The assay mixture contains 400 mM tris hydrochloride pH 8.0, 25 mM[2-¹³C]-sodium pyruvate, 50 mM D,L-glyceraldehyde 3-phosphate, 10 mMMgCl₂, 2 mM thiamine pyrophosphate, 1 mM dithiothreitol, 0.5 mM EDTA,10% D₂O and 0.8 mg enzyme sample in a total volume of 0.5 ml. Themixture is incubated 3 h at 37° C. Protein is precipitated by theaddition of 0.1 ml 50% trichloroacetic acid (TCA). After centrifugationa ¹³C-NMR-spectrum (62.9 Mhz, Bruker, Karlsruhe, Germany) is recorded.The turnover is calculated by integration of the 2C-signals of pyruvateand 1-deoxy-D-xylulose 5-phosphate. Pyruvate displays a 2C-signal at196.5 ppm and a signal at 92.7 ppm which is assigned to thecorresponding hydrate. 1-Deoxy-D-xylulose 5-phosphate displays a signalat 212.5 ppm.

2. By Photometric Detection (Variant A)

The assay mixture contains 200 mM tris hydrochloride pH 8.0, 25 mMsodium pyruvate, 50 mM D,L-glyceraldehyde 3-phosphate (previouslyneutralized with NaOH), 10 mM MgCl₂, 4 mM thiamine pyrophosphate, 8 mMdithiothreitol and 0.02 mg enzyme sample in a total volume of 25 ml. Themixture is incubated 20 min at 37° C. 25 μl of 30% TCA are added. Thesupernatant is added to a buffer containing 200 mM tris hydrochloride pH8.0, 1 mM MnSO₄, 0.5 mM NADPH in a total volume of 0.95 ml. Theextinction at 340 nm is determined. A solution (50 ml, 0.1 U) of1-deoxy-D-xylulose 5-phosphate reductoisomerase is added and the mixtureis incubated 30 min at 37° C. The extinction at 340 nm is determinedagain. The extinction difference is equivalent to the amount of consumedNADPH (ε₃₄₀=6300 M⁻¹cm⁻¹) which is equivalent to the amount of1-deoxy-D-xylulose 5-phosphate produced.

3. By Photometric Detection (Variant B)

The assay mixture contains 200 mM tris hydrochloride pH 8.0, 5 mM sodiumpyruvate, 10 mM D,L-glyceraldehyde 3-phosphate, 1 mM MnSO₄, 1 mMthiamine pyrophosphate, 1 mM dithiothreitol, 0.5 mM NADPH and 1 U of1-deoxy-D-xylulose 5-phosphate reductoisomerase in a total volume of 1ml. The mixture is incubated at 37° C. in a thermostated cuvette and theextinction at 340 nm is monitored. The assay is started by the additionof 5 μl enzyme sample. The negative slope of the extinction isequivalent to the rate of the 1-deoxy-D-xylulose 5-phosphate synthasereaction.

Reference Example 5 Production of an Expression Clone and Constructionof an Expression Vector for 1-deoxy-D-xylulose 5-phosphateReductoisomerase of E. coli

The E. coli ORF yaeM (accession no. gb AE000126) from bp position 9887to 11083 is amplified by PCR using chromosomal E. coli DNA as template.Chromosomal DNA from Escherichia coli strain XL1-Blue is isolatedaccording to a method described in reference example 8.

The reaction mixture contained 25 pmol of primer GGAGGATCCATGAAGCAACTCACC, 25 pmol of primer GCGCGACTCTCTGCAGCCGG, 20 ng ofchromosomal DNA, 2 U of Taq DNA polymerase (Eurogentec, Seraing,Belgium) and 20 nmol of dNTPs in a total volume of 100 μl of 1.5 mMMgCl₂, 50 mM KCl, 10 mM tris hydrochloride, pH 8.8 and 0.1% (w/w) TritonX-100.

The mixture is denaturated for 3 min at 94° C. Then, 30 PCR cycles for45 sec at 94° C., 45 sec at 50° C. and 75 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificates are purified with a PCR purification kit fromQiagen as described in reference example 1.

2.5 μg of the vector pQE30 (Qiagen), isolated as described in referenceexample 1, and 2.0 μg of the purified PCR product are digested in orderto produce DNA fragments with overlapping ends. Each restriction mixturecontains 7 μl of NEB3 buffer, 50 U of BamHI (NEB), 40 U of PstI (NEB) ina total volume of 70 μl and is incubated for 3 h at 37° C. Digestedvector DNA and PCR product are purified using the PCR purification kitfrom Qiagen as described in reference example 1.

20 ng of vector DNA and 22 ng of PCR product are ligated together with 1U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pQEyaeM. The ligation mixture isincubated overnight at 4° C. Each 2 μl of the ligation mixture is usedfor transforming electrocompetent E. coli XL1-Blue and M15-[pREP4](Zamenhof et al., J. Bacteriol. 110, 171-178 (1972)) cells as describedin reference example 1. The electrocompetent cells are prepared asdescribed in reference example 1.

12 μg DNA of plasmid pQEyaeM are obtained.

The DNA insert of the plasmid pQEyaeM is sequenced as described inreference example 1 and is identical with the sequence in the database(accession no. gb AE000126).

Reference Example 6 Preparation and Purification of Recombinant1-deoxy-D-xylulose 5-phosphate Reductoisomerase of E. coli.

Recombinant M15[pREP4] cells of E. coli containing overexpressed1-deoxy-D-xylulose 5-phosphate reductoisomerase of E. coli are preparedidentically to the preparation of reference example 9. The cells arethawed in 20 ml of 20 mM imidazole in 100 mM tris hydrochloride pH 8.0and 0.5 M sodium chloride (standard buffer) in the presence of 1 mg/mllysozyme and 100 μg/ml DNasel. The mixture is incubated at 37° C. for 30min, cooled on ice and sonified 6×10 sec with a Branson Sonifier 250(Branson SONIC Power Company) set to 70% duty cycle output, controlvalue of 4 output. The suspension is centrifuged at 15,000 rpm at 4° C.for 30 min. The cell free extract of recombinant 1-deoxy-D-xylulose5-phosphate reductoisomerase of E. coli is applied on a column ofNi²⁺-chelating sepharose FF (column volume 25 ml, Amersham PharmaciaBiotech) previously equilibrated with 20 mM imidazole in standardbuffer. The column is washed with 100 ml of starting buffer.1-Deoxy-D-xylulose 5-phosphate reductoisomerase is eluted with a lineargradient of 20-500 mM imidazole in standard buffer. 1-Deoxy-D-xylulose5-phosphate reductoisomerase containing fractions are combined accordingto SDS-PAGE and dialysed overnight against 100 mM tris hydrochloride pH8.0. The dialysed 1-deoxy-D-xylulose 5-phosphate reductoisomerase isconcentrated by ultrafiltration (MWCO 10 kDa, Amicon, USA.) and appliedon a Superdex 75 HR 26/60 column (Amersham Pharmacia Biotech). Thehomogeneity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase is judgedby SDS-PAGE. One band at 43 kDa is visible, which is in line with thecalculated molecular mass. The yield of pure 1-deoxy-D-xylulose5-phosphate reductoisomerase is 60 mg.

Reference Example 7 Determination of 1-deoxy-D-xylulose 5-phosphateReductoisomerase Activity

Assay mixtures contain 100 mM tris hydrochloride pH 8.0, 1 mM MnCl₂, 0.5mM NADPH and 5 μg enzyme sample in a total volume of 1 ml. The mixtureis incubated at 37° C. in a thermostated cuvette and the reaction ismonitored spectrophotometrically at 340 nm. The assay is started by theaddition of 10 μl of 50 mM 1-deoxy-D-xylulose 5-phosphate. The negativeslope of the extinction is equivalent to the rate of the reactioncatalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

Reference Example 8 Production of an Expression Clone and Constructionof an Expression Vector for ygbP of E. coli

Cells of Escherichia coli XL1-Blue harboring the expression vectorPNCO113, are grown overnight in Luria Bertani (LB) medium containing 180mg/l of ampicillin for maintenance of the plasmid in the host cells. 7ml of the culture are centrifuged for 20 min at 5,000 rpm. The cellpellet is used for isolation of the plasmid pNCO113 with the miniplasmid isolation kit from Qiagen (Hilden, Germany). The pellet isresuspended in 0.3 ml of 10 mM EDTA in 50 mM tris hydrochloride, pH 8.0.30 μg RNase are added. 0.3 ml of 1% (w/v) SDS in 200 mM sodium hydroxideare added and incubated for 5 min at room temperature. 0.3 ml of chilled3.0 M sodium acetate, pH 5.5 are added and incubated for 10 min on ice.The mixture is centrifuged for 15 min at 14,000 rpm in a minifuge. Thesupernatant is applied onto a Qiagen-tip 20, which is previouslyequilibrated with 1 ml of 750 mM NaCl, 15% (v/v) ethanol and 0.15% (v/v)Triton X-100 in 50 mM MOPS, pH 7.0. The Qiagen-tip is washed four timeswith 1 ml of 1000 mM NaCl and 15% (v/v) ethanol in 50 mM MOPS, pH 7.0.The DNA is eluted with 0.8 ml of 1250 mM NaCl and 15% (v/v) ethanol in50 mM tris hydrochloride, pH 8.5. The DNA is precipitated with 0.56 mlof isopropanol, centrifuged for 30 min at 14,000 rpm and washed with 1ml of ice-cold 70% (v/v) ethanol. After drying in a speedvac for 5 min,the DNA is dissolved in 50 μl of redistilled H₂O. The solution contained8.3 μg of DNA.

Chromosomal DNA from Escherichia coli strain XL1-Blue is isolatedaccording to a method described by Meade, et al. (J. Bacteriol. 149,114-122 (1982)). The E. coli ORF ygbP (accession no. gb AE000358) frombasepair (bp) position 6754 to 7464 is amplified by PCR usingchromosomal E. coli DNA as template. The reaction mixture contained 25pmol of primer AAATTAACCATGGCAACCACTCAT TTGG, 25 pmol of primerTTGGGCCTGCAGCGCCAAAGG, 20 ng of chromosomal DNA, 2 U of Taq DNApolymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in 1.5 mMMgCl₂, 50 mM KCl, 10 mM tris hydrochloride pH 8.8 and 0.1% (w/w) TritonX-100 in a total volume of 100 μl.

The mixture is denaturated for 3 min at 95° C. Then 25 PCR cycles for 30sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis. The PCRamplificate is purified with the PCR purification kit from Qiagen. 500μl of buffer PB (Qiagen) are added to 98 μl of PCR reaction mixture andapplied to a Qiaquick column and centrifuged for 1 min at 14,000 rpm.The flow through is discarded. 0.75 ml of buffer PE (Qiagen) are loadedon the column and centrifuged as before. The flow through is discardedand the column is centrifuged for an additional 1 min at 14,000 rpm. Thecolumn is placed in a clean 1.5 ml eppendorf tube. 50 μl of H₂O(redistilled, sterile) are added to the column and it is centrifuged for1 min at 14,000 rpm. The flow through contained 1.5 μg of purified PCRproduct.

2.0 μg of the vector pNCO113 and 1.5 μg of the purified PCR product aredigested in order to produce DNA fragments with overlapping ends. Eachrestriction mixture contained 7 μl of NEB3 buffer (NEB), 7 μg of BSA, 40U of NcoI (NEB), 30 U of PstI (NEB) in a total volume of 70 μl and isincubated for 3 h at 37° C. Digested vector DNA and PCR product arepurified using the PCR purification kit from Qiagen.

20 ng of vector DNA and 16 ng of PCR product are ligated together with 1U of T4-Ligase from Gibco-BRL (Eggenstein, Germany), 2 μl of T4-Ligasebuffer (Gibco) in a total volume of 10 μl yielding the plasmid pNCOygbP.The ligation mixture is incubated overnight at 4° C. With 2 μl of theligation mixture electrocompetent E. coli XL1-Blue cells aretransformed.

The plasmid pNCOygbP is isolated as described in reference example 1.

The DNA insert of the plasmid pNCOygbP is sequenced as described inreference example 1.

Reference Example 9 Preparation and Purification of Recombinant YgbPProtein of E. coli

0.5 liter of Luria Bertani (LB) medium containing 90 mg of ampicillinare inoculated with 10 ml of an overnight culture of E. coli strainXL1-Blue harboring plasmid pNCOygbP. The culture is grown in a shakingculture at 37° C. At an optical density (600 nm) of 0.7, the culture isinduced with 2 mM IPTG. The culture is grown for further 5 h. The cellsare harvested by centrifugation for 20 min at 5,000 rpm and 4° C. Thecells are washed with 50 mM tris hydrochloride pH 8.0, centrifuged asabove and frozen at −20° C. for storage.

The cells are thawed in 10 ml of 20 mM tris hydrochloride pH 8.0containing 1 mM dithioerythritol, 0.02% sodium azide (buffer A) in thepresence of 4 mg/ml lysozyme and 10 μg/ml DNasel. The mixture isincubated at 37° C. for 1 h, cooled on ice and sonified 6×10 sec with aBranson Sonifier 250 (Branson SONIC Power Company, Danbury, USA) set to70% duty cycle output, control value of 4 output. The suspension iscentrifuged at 15,000 rpm at 4° C. for 30 min. The supernatant isapplied on a column of Sepharose Q FF (size 4.6×24 cm, AmershamPharmacia Biotech, Freiburg, Germany) previously equilibrated with 200ml buffer A. The column is washed with buffer A monitoring at 280 nm.4-Diphosphocytidyl-2C-methyl-D-erythritol synthase is eluted from thecolumn with a gradient from 0-0.5 M sodium chloride in 300 ml of bufferA. The enzyme is identified by SDS-PAGE showing a band at 26 kDa.Fractions showing this protein band are collected and dialysed againstbuffer A overnight. The enzyme is further purified on a column of RedSepharose CL-6B (size 2.6×10 cm, Amersham Pharmacia Biotech)equilibrated with buffer A. The enzyme is passed through the column andis loaded on a Source 15Q (column volume 20 ml, Amersham PharmaciaBiotech). The enzyme is eluted by a gradient of 0-0.5 M sodium chloridein 250 ml buffer A. The homogeneity of4-diphosphocytidyl-2C-methyl-D-erythritol synthase is judged bySDS-PAGE.

Reference Example 10 Enzymatic Production of4-diphosphocytidyl-2C-methyl-D-erythritol

A solution containing 100 mM tris HCl pH 8.0, 10 mM MgCl₂, 10 mM CTP,0.12 μCi of [2-¹⁴C]2C-methyl-D-erythritol 4-phosphate, 46 mM of2C-methylerythritol 4-phosphate and 225 μg of YgbP protein fromrecombinant E. coli is incubated at 37° C. for 1 h. The reaction ismonitored by ³¹P-NMR. The product displaying two ³¹P-NMR dubletts at−7.2 ppm and −7.8 ppm is purified by HPLC on a column of the anionicexchanger Nucleosil 10SB (4.6×250 mm) using 0.1 M ammonium formate in40% (v/v) methanol as eluent at a flow rate of 1 ml/min. The eluent ismonitored by a UV-diode array detector (J&M TIDAS) and a radiomonitorfrom Berthold. 4-Diphosphocytidyl-2C-methyl-D-erythritol is eluted at 30ml. The fraction containing 4-diphosphocytidyl-2C-methyl-D-erythritol iscollected and lyophylized. The residue is dissolved in 0.5 ml ofdeuterated water and subjected to NMR analysis.

Reference Example 11 Production of an Expression Clone and Constructionof an Expression Vector for ychB of E. coli

Chromosomal DNA from E. coli strain XL1-Blue is isolated as described inreference example 8.

The E. coli ORF ychB (accession no. gb AE000219) from basepair (bp)position 5720 to 6571 is amplified by PCR using chromosomal E. coli DNAas template. The reaction mixture contained 25 pmol of primer5′-GAGGAGAAATTAACCATGCGGACACAGTGGCC-3′, 25 pmol of primer5′-GTCACCGAACTGCAGCTTGCCCG-3′, 20 ng of chromosomal DNA, 2 U of Taq DNApolymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in of 1.5mM MgCl₂, 50 mM KCl, 10 mM tris hydrochloride pH 8.8 and 0.1% (w/w)Triton X-100 in a total volume of 100 μl.

The mixture is denaturated for 3 min at 95° C. Then 25 PCR cycles for 30sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis. The PCRamplificate is purified with PCR purification kit from Qiagen. 1.5 μg ofpurified PCR product are obtained.

The PCR amplificate is used as template for a second PCR reaction. Thereaction mixture contained 25 pmol of primer5′-ACACAGAATTCATTAAAGAGGAGAAATTAACCATG-3′, 25 pmol of primerGTCACCGAACTGCAGCTTGCCCG-3′, 2 μl of the first PCR amplification, 2 U ofTaq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPsin a total volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 40 PCR cycles for 45sec at 94° C., 45 sec at 50° C. and 60 sec at 72° C. follow. Afterfurther incubation for 20 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with the PCR purification kit fromQiagen as described in reference example 1.

2.0 μg of the vector pNCO113 and 1.5 μg of the purified PCR product aredigested in order to produce DNA fragments with overlapping ends. Eachrestriction mixture contained 6 μl of NEB3 buffer, 6 μg of BSA, 30 U ofEcoRI (NEB), 30 U of PstI (NEB) in a total volume of 60 μl and isincubated for 3 h at 37° C. Digested vector DNA and PCR product arepurified using the PCR purification kit from Qiagen.

20 ng of vector DNA and 18 ng of PCR product are ligated together with 1U of T4-Ligase from Gibco-BRL (Eggenstein, Germany), 2 μl of T4-Ligasebuffer (Gibco-BRL) in a total volume of 10 μl yielding the plasmidpNCOychB. The ligation mixture is incubated overnight at 4° C. With 2 μlof the ligation mixture electrocompetent E. coli XL1-Blue cells aretransformed and 100 μl of the cell/DNA suspension is plated on LB platescontaining 150 mg/l ampicillin for maintenance of the plasmid pNCOychB.The plasmid pNCOychB is isolated as described before. 9 μg of plasmidDNA are obtained.

The DNA insert of the plasmid pNCOychB is sequenced as described inreference example 1. The DNA sequence is found to be identical to thesequence in the data base (accession no. gb AE000219).

Reference Example 12 Preparation and Purification of Recombinant YchBProtein of E. coli

0.5 liter of LB medium containing 90 mg of ampicillin are inoculatedwith 10 ml of an overnight culture of E. coli strain XL1-Blue harboringplasmid pNCOychB. The culture is grown in a shaking culture at 37° C. Atan optical density (600 nm) of 0.7, the culture is induced with 2 mMIPTG. The culture is grown for further 5 h. The cells are harvested bycentrifugation for 20 min at 5,000 rpm and 4° C. The cells are washedwith 50 mM tris hydrochloride pH 8.0, centrifuged as above and frozen at−20° C. for storage.

The cells are thawed in 10 ml of 20 mM tris hydrochloride pH 8.0containing 1 mM dithioerythritol, 0.02% sodium azide (buffer A) in thepresence of 4 mg/ml lysozyme and 10 μg/ml DNasel. The mixture isincubated at 37° C. for 1 h, cooled on ice and sonified 6×10 sec with aBranson Sonifier 250 (Branson SONIC Power Company, Danbury, USA) set to70% duty cycle output, control value of 4 output. The suspension iscentrifuged at 15,000 rpm at 4° C. for 30 min. The supernatant isapplied on a column of Sepharose QFF (column volume 30 ml, AmershamPharmacia Biotech, Freiburg, Germany) previously equilibrated with 150ml buffer A. The column is washed with buffer A monitoring at 280 nm.YchB protein is eluted from the column with a gradient from 0-0.5 Msodium chloride in 150 ml of buffer A. The enzyme is identified bySDS-PAGE showing a band at 30 kDa. Fractions showing this protein bandare collected and ammonium sulfate is added to a final concentration of0.5 M. The enzyme is further purified on a column of Phenyl Sepharose6FF (column volume 16 ml, Amersham Pharmacia Biotech) equilibrated withbuffer A containing 0.5 M ammonium sulfate. Then the YchB protein iseluted by linear gradient from 0.5-0 M ammonium sulfate in 100 ml ofbuffer A. Fractions containing protein are pooled and concentrated to 3ml by ultrafiltration (MWCO 10 kDa, Amicon, USA). Then the enzyme isfurther purified on Superdex 75 HR 26/60 equilibrated with buffer A inthe presence of 100 mM sodium chloride. The YchB protein is eluted at165 ml. The homogeneity of the YchB protein is judged by SDS-PAGE.

Reference Example 13 Enzymatic Preparation of4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate

A solution containing 5 mM[2-¹⁴C]4-diphosphocytidyl-2C-methyl-D-erythritol (0.04 μCi/mmol), 5 mMATP, 5 mM MgCl₂, 5 mM DTT, 100 μg of purified YchB protein and 100 mMtris hydrochloride pH 8.0 in a total volume of 4 ml is incubated for 2 hat 37° C. The reaction is monitored by ³¹P-NMR spectroscopy. Then thesample is centrifuged through a Nanosep 10K membrane (PALLGelmann,Robdorf, Germany). The product displaying ³¹P signals at 0.49, −7.28,and −8.00 ppm (referenced to external 85% phosphoric acid) is purifiedby HPLC on a column of the anionic exchanger Nucleosil 10SB (4.6×250 mm,Macherey-Nagel, Düren, Germany), equilibrated with 0.1 M ammoniumformate in 40% (v/v) methanol at a flow rate of 1 ml/min. The HPLCsystem is equipped with a Wellchrom HPLC pump K-1001, a WellchromSpectro-Photometer K-2600 (Knauer, Berlin, Germany) and a radiomonitor(Berthold, Wildbad, Germany). After injection of the sample, the columnis washed with 30 ml of 0.1 M ammonium formate in 40% (v/v) methanol.4-Diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate is eluted at 14 mlby a linear gradient from 0.1 M ammonium formate in 40% (v/v) methanolto 1 M ammonium formate in 0% (v/v) methanol in 30 ml. Fractionscontaining 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate arecollected and lyophylized. The residue is dissolved in 0.5 ml ofdeuterated water and subjected to NMR analysis. The concentration of4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate is 21 mM.

Reference Example 14 Construction of an Expression Clone for ygbB fromE. coli

The E. coli ORF ygbB (accession no. gb AE000358) from bp position 6231to 6754 is amplified by PCR using chromosomal E. coli DNA as template.Chromosomal DNA from E. coli strain XL1-Blue is isolated as described inreference example 8.

The reaction mixture contained 10 pmol of primer GAGGAGAAATTAACCATGCGAATTGGACACGGTTTTG, 10 pmol of primer TATTATCTGCAGCCTTGCGGTTTACCGTGGAGG, 20 ng of chromosomal DNA, 2 U of Taq DNA polymerase(Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in a total volume of100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM tris hydrochloride, pH 8.8 and0.1% (w/w) Triton X-100.

The mixture is denaturated for 5 min at 94° C. Then 30 PCR cycles for 30sec at 94° C., 45 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis. The PCRamplificate is used as template for a second PCR reaction. The reactionmixture contained 50 pmol of primer ACACAGAATTCATTAAAGAGGAGAAATTAACCATG,50 pmol of primer TATTATCTGCAGCCTTGCGGTTTACCGTGGAGG, 2.5 μl of the firstPCR amplification, 10 U of Taq DNA polymerase (Eurogentec, Seraing,Belgium) and 100 nmol of dNTPs in a total volume of 500 μl of 1.5 mMMgCl₂, 50 mM KCl, 10 mM tris hydrochloride, pH 8.8 and 0.1% (w/w) TritonX-100. The mixture is apportioned in 5 PCR-tubes.

The mixtures are denaturated for 5 min at 94° C. Then 25 PCR cycles for30 sec at 94° C., 45 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to a agarose gel electrophoresis.

The PCR amplificates are purified with a PCR purification kit fromQiagen as described in reference example 1.

4.5 μg of the vector pNCO113 (isolated as described in referenceexample 1) and 3.4 μg of the purified PCR product are digested in orderto produce DNA fragments with overlapping ends. Each restriction mixturecontains 20 μl of NEB3 buffer, 100 U of EcoRI (NEB), 100 U of PstI (NEB)in a total volume of 200 μl and is incubated for 3 h at 37° C. Digestedvector DNA and PCR product are purified using the PCR purification kitfrom Qiagen as described in reference example 1.

100 ng of vector DNA and 35 ng of PCR product are ligated together with1 U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pNCOygbB. The ligation mixture isincubated for 2 h at 25° C. 1 μl of the ligation mixture is transformedinto electrocompetent E. coli XL1-Blue cells as described in referenceexample 1. The electrocompetent cells are prepared as described inreference example 1.

Reference Example 15 Preparation and Purification of Recombinant YgbBProtein of E. coli

The cell free extract of YgbB protein from E. coli is preparedidentically to the preparation in reference example 9. The supernatantis applied on a column of Sepharose Q FF (column volume 30 ml, AmershamPharmacia Biotech, Freiburg, Germany) previously equilibrated with 120ml of buffer A. The column is washed with 90 ml of buffer A. Then theYgbB protein is eluted with a linear gradient of 0-0.5 M NaCl in 150 mlbuffer A. The homogeneity of YgbB protein is judged by SDS-PAGE.

Reference Example 16 Production of an Expression Clone and Constructionof an Expression Vector for a 6× His-YgbB Fusion Protein of E. coli

The E. coli ORF ygbB (accession no. gb AE000358) from bp position 6231to 6754 is amplified by PCR using chromosomal E. coli DNA as template.Chromosomal DNA from E. coli strain XL1-Blue is isolated according to amethod described in reference example 8.

The reaction mixture contained 10 pmol of primerGAGAAGGATCCATGCGAATTGGACACGGTTTTGACG, 10 pmol of primerTATTATCTGCAGCCTTGCGGTTTACCGTGGAGG, 20 ng of chromosomal DNA, 2 U of TaqDNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in atotal volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 5 min at 94° C. Then 30 PCR cycles for 30sec at 94° C., 45 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with a PCR purification kit from Qiagenas described in reference example 1.

1.0 μg of the vector pQE30, isolated as described in reference example 1(Qiagen) and 0.5 μg of the purified PCR product are digested in order toproduce DNA fragments with overlapping ends. Each restriction mixturecontains 10 μl of NEB3 buffer from NEB, 100 U of BamHI (NEB), 100 U ofPstI (NEB) in a total volume of 100 μl and is incubated for 3 h at 37°C. Digested vector DNA and PCR product are purified using the PCRpurification kit from Qiagen as described in reference example 1.

5 fmol of vector DNA and 14 fmol of PCR product are ligated togetherwith 1 U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in atotal volume of 10 μl, yielding the plasmid pQEygbB. The ligationmixture is incubated for 2 h at 25° C. 1 μl of the ligation mixture istransformed into electrocompetent E. coli XL1-Blue cells as described inreference example 1. The electrocompetent cells are prepared asdescribed in reference example 1. The plasmid pQEygbB is isolated asdescribed in reference example 1.12 μg of plasmid DNA are obtained.

The DNA insert of the plasmid pQEygbB is sequenced as described inreference example 1 and is identical to the sequence in the database(accession no. gb AE000358). The 5′-end of the DNA insert carries thecoding region for 6 histidine residues.

Reference Example 17 Preparation and Purification of Recombinant 6×His-YgbB Fusion Protein of E. coli

Recombinant XL1-Blue cells of E. coli containing overexpressed YgbB(N-terminal His-tagged) of E. coli are prepared as in reference example9. The cells are thawed in 20 ml of 20 mM imidazole in 100 mM trishydrochloride pH 8.0 and 0.5 M sodium chloride (standard buffer) in thepresence of 1 mg/ml lysozyme and 100 μg/ml DNasel. The mixture isincubated at 37° C. for 30 min, cooled on ice and sonified 6×10 sec witha Branson Sonifier 250 (Branson SONIC Power Company) set to 70% dutycycle output, control value of 4 output. The suspension is centrifugedat 15,000 rpm at 4° C. for 30 min. The cell free extract of recombinantYgbB protein is applied on a column of Ni²⁺-Chelating sepharose FF(column volume 25 ml, Amersham Pharmacia Biotech) previouslyequilibrated with 20 mM imidazole in standard buffer. The column iswashed with 100 ml of starting buffer. YgbB protein is eluted with alinear gradient of 20-500 mM imidazole in standard buffer. YgbBprotein-containing fractions are combined according to SDS-PAGE anddialysed overnight against 100 mM tris hydrochloride pH 8.0. Thedialysed YgbB protein is concentrated by ultrafiltration (MWCO 10 kDa,Amicon, USA.) and applied on Superdex 75 HR 26/60 (Amersham PharmaciaBiotech). The homogeneity of YgbB protein is judged by SDS-PAGE. Theobjected band at 17 kDa is in agreement with the calculated molecularmass. 27 mg of pure enzyme were obtained.

Reference Example 18 Enzymatic Preparation of 2C-methyl-D-erythritol2,4-cyclopyrophosphate

A solution containing 5 mg[2-¹⁴C]4-diphosphocytidyl-2C-methyl-D-erythritol (0.02 μCi/mmol), 5 mMMgCl₂, 5 mM ATP, 5 mM DDT, 100 μg purified YchB protein, 200 μg purifiedYgbB protein and 100 mM tris hydrochloride pH 8.0 in a total volume of 4ml is incubated for 2 h at 37° C. The reaction is monitored by ¹³C- and³¹P-NMR spectroscopy. The solution is passed through a Nanosep 10Kmembrane (PALLGemann, Robdorf, Germany). The product displaying two³¹P-NMR signals at −7.65 ppm and −11.66 ppm (dubletts, ³¹P—³¹P couplingconstant, 23.6 Hz) and displaying two intense ¹³C-NMR signals at 83.87ppm is purified by HPLC on a column of the anionic exchanger Nucleosil10SB (4.6×250 mm, Macherey-Nagel, Düren, Germany) using 40% (v/v)methanol containing 0.1 M ammonium formate as eluent at a flow rate of 1ml/min. 2C-methyl-D-erythritol 2,4-cyclopyrophosphate is eluted at 34ml. Fractions containing 2C-methyl-D-erythritol 2,4-cyclopyrophosphateare collected and lyophylized. The residue is dissolved in 0.5 ml ofdeuterated water and subjected to NMR analysis. The concentration of2C-methyl-D-erythritol 2,4-cyclopyrophosphate is 18 mM.

Reference Example 19 Identification of 2C-methyl-D-erythritol2,4-cyclopyrophosphate

The elucidation of the structure is performed with[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate (Table6).

¹H-NMR and ¹H-decoupled ¹³C-NMR spectra are recorded using a AVANCE DRX500 spectrometer from Bruker (Karlsruhe, Germany). The frequencies are500.1 MHZ and 125.6 Mhz for ¹H and ¹³C, respectively. The chemicalshifts are referenced to external trimethylsilylpropane sulfonate.³¹P-NMR spectra are recorded using a AC 250 spectrometer from Bruker ata frequency of 101.3 MHz. The chemical shifts are referenced to external85% H₃PO₄.

The structure of the product is evaluated by a multinuclearmultidimensional NMR approach (Table 6). Specifically, the compound ischaracterized by two ¹H decoupled ³¹P-NMR signals at −7.65 ppm (dublettwith a ³¹P—³¹P coupling constant of 23.6 Hz) and −11.66 ppm(double-double dublett with a ³¹P—³¹P coupling constant of 23.6 Hz and a³¹P—¹³C coupling constant of 8.5 Hz, respectively). The ³¹P-NMR signalat 7.65 ppm is broadened without ¹H decoupling. The detected ³¹P-NMRchemical shift range, as well as the ³¹P—³¹P couplings implied that theunknown compound is a pyrophosphate. Moreover, the detected ³¹P—¹³Ccouplings for the ³¹P-NMR signal at −11.66 ppm in conjunction with themissing ³¹P—¹H coupling for the signal indicate that one phosphate unitof the pyrophosphate moiety is connected to C-2 of2C-methyl-D-erythritol. In line with this conclusion, ¹³C—³¹P couplingsare observed for the ¹³C-NMR signals reflecting C-2 and C-2-methyl.

In conjunction with the observed ¹³C—¹³C couplings (Table 6), these dataare the basis of the ¹H- and ¹³C-NMR signal assignments. The ¹³C signalat 65.72 ppm (reflecting C-4) showed ¹³C—³¹P coupling suggesting thatthe pyrophosphate motif is also connected to C-4. The ¹³C-NMRassignments are further confirmed by two-dimensional INADEQUATEexperiments establishing the ¹³C—¹³C connectivities.

In summary, the ¹H-, ¹³C- and ³¹P-NMR data clearly established theproduct as 2C-methyl-D-erythritol 2,4-cyclopyrophosphate. The NMR datawere in close correspondence to reported data for this compound(Ostrovsky et al. Biofactors 4 (1), 63-68 (1992); Turner et al. Biochem.J. 285, 387-390 (1992)).

TABLE 6 NMR data of [2,2-methyl-¹³C₂]-2C-methyl- D-erythritol2,4-cyclopyrophosphate Chemical shifts, ppm Coupling constants, HzPosition ¹H ¹³C ³¹P J_(PH) J_(PC) J_(PP) J_(CH) J_(CC) J_(HH) 1 3.51(dt, 66.95 (d)^(b) 1.7 41.8 (2) 12.4 (1) 1H)^(a)  1* 3.66 (dd, 1.8 12.4(1) 1H) 2 83.87 (dd) 8.4 39.8 (2-methyl) 2- 1.31 (dd, 16.30 (dd) 5.3128.4, 39.8 (2) methyl 3H) 4.0 3 3.98 68.42 (dm) n.d. 46.0 (2) n.d 465.72 (d) 6.6 n.d.  4* 4.13 (m, n.d. 3H) P (4)  −7.65 (d)^(c) n.d. 23.6P (2) −11.66 (ddd) 8.5, 5.3 23.6 ^(a)Referenced to externaltrimethylsilylpropane sulfonate. The multiplicities and the relativeintegral values of signals in the ¹H-NMR spectrum are given inparentheses. ^(b)Referenced to external trimethylsilylpropane sulfonate.The multiplicities of the ¹H decoupled ¹³C-NMR signals are given inparentheses. ^(c)Referenced to external 85% ortho-phosphoric acid. Themultiplicities of the ¹H decoupled ³¹P-NMR signals are given inparentheses.

Reference Example 20 Comprehensive Enzymatic Synthesis of4-diphosphocytidyl-[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol Step a)Enzymatic Synthesis of [1,2-¹³C₂]-1-deoxy-D-xylulose 5-phosphate

Crude dihydroxyacetone phosphate is prepared as described by Effenbergerand Straub (Tetrahedron Lett. 28, 1641-1644 (1987)). 1 g ofdihydroxyacetone phosphate is dissolved in 70 ml of a solution of 57 mM[2,3-¹³C₂]sodium pyruvate, 10 mM MgSO₄ and 2.5 mM thiaminepyrophosphatein 150 mM tris hydrochloride, pH 8.0. 17,000 U of triose phosphateisomerase (rabbit muscle) are added and the solution is incubated 105min at 37° C. 0.774 ml (7.4 U) of recombinant 1-deoxyxylulose5-phosphate synthase from B. subtilis are added. The reaction ismonitored as described in reference example 4. After 8 h the reaction isstopped by adjusting the pH to a value of 3 by addition of 1 M HCl (11.2ml). The reaction mixture is stored at −20° C.

Step b) Enzymatic Synthesis of [2,2-methyl-¹³C₂]-2C-methyl-D-erythritol4-phosphate

To the reaction mixture obtained in step a, containing[1,2-¹³C₂]-1-deoxy-D-xylulose 5-phosphate, 19 ml of 1 M tris buffer pH8.0, 1.1 ml of 1 M MgCl₂ solution, 3 g glucose (72 mmol) and 6 ml ofsolution of 0.1 M MnCl₂ are added and the pH is adjusted to 8.0 with (7ml 1 M NaOH). Precipitate is separated by centrifugation. To a finalvolume of 200 ml, water, 250 U of glucose dehydrogenase from B.megaterium and 56.6 mg NADP⁺ (80 μmol) are added. After 5 min ofpreincubation at 37° C., 2 ml (11.2 U) of recombinant 1-deoxy-D-xylulose5-phosphate reductoisomerase from E. coli are added. After ca. 30 h thereaction is stopped by the addition of 8 ml of 2 N HCl. The reactionmixture is stored at −20° C.

Step c) Enzymatic Synthesis of4-diphosphocytidyl-[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol

The pH of the reaction mixture obtained in step b, containing[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol 4-phosphate, is adjusted to 7.0by addition of 4 ml 2 M NaOH. 1.4 g of CTP (2.5 mmol) is added and thepH is adjusted to 8.0 with 6 ml 2 N NaOH. After 5 min of preincubationat 37° C., 1.5 ml (51.8 U) of YgbP protein from E. coli solution areadded. The reaction is monitored as described in reference example 10.After ca. 5 h the reaction mixture is purified and lyophylized asdescribed in reference example 10. 550 mg of pure4-diphosphocytidyl-[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol areobtained.

Reference Example 21 Enzymatic Synthesis of4-diphosphocytidyl-[2,2-methyl-¹³C₂]-2C-methyl-D-erythritol in aOne-Vial Reaction

A reaction mixture containing 3 g glucose, 1 g of dihydroxyacetonephosphate (5.7 mmol), 1.4 g of CTP (2.5 mmol), 0.45 g of 2,3-¹³C₂-sodiumpyruvate (4 mmol), 56.6 mg NADP⁺ (80 μmol), in 150 mM trishydrochloride, pH 8.0 is prepared. 17,000 U of triose phosphateisomerase, 250 U of glucose dehydrogenase, 7 U of 1-deoxyxylulose5-phosphate synthase, 13 U of 1-deoxy-D-xylulose 5-phosphatereductoisomerase and 55 U of YgbP protein are added. To a final volumeof 200 ml, 10 mM MgCl₂, 10 mM MnSO₄, 2.5 mM thiamine pyrophosphate in150 mM tris hydrochloride, pH 8.0 are added. The pH is adjusted to 8.0with 5 ml 1 M NaOH. The reaction mixture is incubated at 37° C. Thereaction is monitored as described in reference example 10. After 30 hthe reaction mixture is purified and lyophylized as described inreference example 10.490 mg of pure4-diphosphocytidyl-[2,2-methyl-¹³C₂]-methyl-D-erythritol are obtained.

Reference Example 22 Large Scale Preparation of 2C-methyl-D-erythritol4-phosphate

This preparation can be performed with any ¹³C-labeled sample of glucoseor pyruvate as starting materials. In this example, it is described for[U—¹³C₆]-glucose and [2,3-¹³C₂]-pyruvate.

Step a) Preparative Synthesis of [U—¹³C₅]-1-deoxy-D-xylulose 5-phosphate

A reaction mixture containing 166 mg [U—¹³C₆]-glucose (0.89 mmol), 44 mgthiamine pyrophosphate, 1.02 g of ATP (1.79 mmol), 200 mg of[2,3-¹³C₂]-pyruvate (1.79 mmol), 6 mM MgCl₂ in 150 mM trishydrochloride, pH 8.0 is prepared. 410 U of triose phosphate isomerase(from rabbit muscle, Type III-S, E. C. 5.3.1.1, Sigma), 360 U hexokinase(from Bakers Yeast, Type VI, E. C. 2.7.1.1, Sigma), 50 U phosphoglucoseisomerase (from Bakers Yeast, Type III, E. C. 5.3.1.9, Sigma), 20 Uphosphofructokinase (from Bacillus stearothermophilus, Type VII, E. C.2.7.1.11, Sigma), 35 U aldolase (from rabbit muscle, E C. 4.1.2.13,Sigma) and 2 U recombinant DXP synthase from B. subtilis are added to afinal volume of 58 ml. The reaction mixture is incubated at 37° C.overnight. During the reaction, the pH is held at a constant value of8.0 by the addition of 1 M NaOH (2 ml). The reaction is stopped byadding 3 ml of 2 N hydrochloric acid. ¹³C-NMR-spectra are recorded formonitoring the conversion (Table 7).

TABLE 7 NMR data of [U-¹³C₅]-1-deoxy-D-xylulose 5-phosphate Chemicalshifts, ppm^(a) Coupling constants, Hz Position ¹³C J_(PC) J_(CC) 1 25.941.1 (2), 12.8 (3) 2 213.1 41.3 (1), 41.3 (3), 3.1 (5) 3 77.0 41.5 (2),40.2 (4), 12.8 (1) 4 70.7 6.9 43.2 (5), 39.6 (3) 5 64.3 4.6 43.4 (4),3.1 (2) ^(a)Referenced to external trimethylsilylpropane sulfonate.

Step b) Preparative Synthesis of [U—¹³C₅]-2C-methyl-D-erythritol4-phosphate

To the solution of step a) 10 U DXP reductoisomerase, 120 U glucosedehydrogenase (from Bacillus megaterium, E. C. 1.1.1.47, Sigma), 0.97 gglucose, 200 mM MgCl₂ and 0.3 mM NADP⁺ are added. The pH is adjusted to8.0 with 1.5 ml of 4 N sodium hydroxide. After centrifugation the volumeis 72 ml. The reaction mixture is incubated at 37° C. overnight. Theconversion is monitored by recording ¹³C-NMR-spectra of the accumulatingproduct (Table 8). The reaction product is purified by HPLC on a columnof the anionic exchanger Nucleosil 10 SB (16×250 mm) using 0.5 M formicacid as eluent at a flow rate of 13 ml/min. The eluent is monitored by arefractometer (GAT-LCD210 from Gamma Analyse Technik, Bremerhafen,Germany). The product is eluted at 14.5 min. The fraction containing[U—¹³C₅]-2C-methyl-D-erythritol 4-phosphate is collected andlyophylized. The amount is 86 mg.

TABLE 8 NMR data of [U-¹³C₅]-2C-methyl-D-erythritol Position Chemicalshifts, ppm^(a) 1 66.5 2 74.1 2-Methyl 18.5 3 74.1 4 64.6 ^(a)Referencedto external trimethylsilylpropane sulfonate.

Reference Example 23 Enzymatic Synthesis of4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate

This preparation can be performed with any ¹³C-labeled sample of2C-methyl-D-erythritol 4-phosphate as starting material. In this exampleit is described for [1,3,4-¹³C₃]-2C-methyl-D-erythritol 4-phosphate.

To a reaction mixture containing 15 mg of purified[1,3,4-¹³C₃]-2C-methyl-D-erythritol 4-phosphate (69 μmol), 34 mg CTP (69μmol), 16 mg sodium phosphoenol pyruvate (69 μmol), 1.9 mg ATP (3.5μmol), 10 mg MgCl₂, 5 mM DTT, 10 mM KCl and 150 mM tris hydrochloride,pH 8.0, 60 μl of YgbP protein (2.1 mg/ml), 200 μl of YchB protein (0.3mg/ml) and 100 U of pyruvate kinase (from rabbit muscle, Type VII, E. C.2.7.1.40, Sigma) are added. The final volume is 5 ml. The reactionmixture is incubated at 37° C. for 4 h. The reaction is monitored asdescribed in reference example 13.

4-diphosphocytidyl-methyl-D-erythritol 2-phosphate is purified by HPLCon a column of the anionic exchanger Nucleosil 5 SB (7.5×150 mm) using agradient of 1 M ammonium formiate (B) and 100 mM ammonium formiate (A)as eluent at a flow rate of 3.1 ml/min.

t (min) A (%) B (%) 0 100 0 5 100 0 35 30 70 40 30 70

The eluent is monitored by a UV-detector (Knauer) at 275 nm.4-diphosphocytidyl-methyl-D-erythritol 2-phosphate is eluted at 26-27min.

Example 1 Construction of an Expression Vector and Production of anExpression Clone for the gcpE Gene of Escherichia coli

Cells of Escherichia coli XL1-Blue (Bullock et al. BioTechniques 5,376-379 (1987); commercial source: Stratagene, LaJolla, Calif., USA)harboring the vector pNCO113 (German patent application 19948887.8), aregrown overnight in Luria Bertani (LB) medium containing 180 mg/l ofampicillin for maintenance of the plasmid in the host cells. 7 ml of theculture are centrifuged for 20 min at 5,000 rpm. The cell pellet is usedfor isolation of the plasmid pNCO113 with the mini plasmid isolation kitfrom Qiagen. The pellet is resuspended in 0.3 ml of 10 mM EDTA in 50 mMtris hydrochloride, pH 8.0. 30 μg RNase A are added. 0.3 ml of 1% (w/v)SDS in 200 mM sodium hydroxide are added and incubated for 5 min at roomtemperature. 0.3 ml of chilled 3.0 M sodium acetate, pH 5.5 are addedand incubated for 10 min on ice. The mixture is centrifuged for 15 minat 14,000 rpm in a minifuge. The supernatant is applied onto aQiagen-tip 20, which is previously equilibrated with 1 ml of 750 mMNaCl, 15% (v/v) ethanol and 0.15% (v/v) Triton X-100 in 50 mM MOPS, pH7.0. The Qiagen-tip is washed four times with 1 ml of 1000 mM NaCl and15% (v/v) ethanol in 50 mM MOPS, pH 7.0. The DNA is eluted with 0.8 mlof 1250 mM NaCl and 15% (v/v) ethanol in 50 mM tris hydrochloride, pH8.5. The DNA is precipitated with 0.56 ml of isopropanol, centrifuged 30min at 14,000 rpm and washed with 1 ml of ice-cold 70% (v/v) ethanol.After drying in a speedvac for 5 min, the DNA is dissolved in 50 μl ofredistilled H₂O. The solution contains 7 μg of the vector DNA pNCO113.

The E. coli ORF gcpE (accession no. gb AE000338) is amplified from basepair (bp) position 372 to 1204 by PCR using chromosomal E. coli DNA astemplate. The reaction mixture contains 25 pmol of the primer5′-GAGGAGAAATTAACCATGCATAACCAGGCTCC-3′, 25 pmol of the primer5′-CGAGGCGGATCCCATCACG-3′, 20 ng of chromosomal DNA, 2 U of Taq DNApolymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in atotal volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 45sec at 94° C., 45 sec at 50° C. and 60 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with the PCR purification kit fromQiagen (Hilden, Germany). To 100 μl of PCR reaction mixture 300 μl ofQX1 buffer are added and the mixture is applied to a Qiaquick spincolumn and centrifuged for 1 min at 14,000 rpm. The flow through isdiscarded. 0.75 ml of buffer PE (Qiagen) are loaded on the column andcentrifuged as before. The flow through is discarded and the column iscentrifuged for an additional 1 min at 14,000 rpm. The column is placedin a clean 1.5 ml eppendorf tube. 50 μl of H₂O (redistilled, sterile)are added to the column and it is centrifuged for 1 min at 14,000 rpm.The flow through contains 1.2 μg of purified PCR product.

The PCR amplificate is used as template for a second PCR reaction. Thereaction mixture contains 25 pmol of the primer5′-ACACAGAATTCATTAAAGAGGAGAAATTAACCATG-3′, 25 pmol of the primer5′-CGAGGCGGATCCCATCACG-3′, 2 μl of the first PCR amplification, 2 U ofTaq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPsin a total volume of 100 μl containing 1.5 mM MgCl₂, 50 mM KCl, 10 mMtris hydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 45sec at 94° C., 45 sec at 50° C. and 60 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with the PCR purification kit fromQiagen as described above. 1.1 μg of purified PCR product are obtained.2.0 μg of the vector pNCO113 and 1.1 μg of the purified PCR product aredigested in order to produce DNA fragments with overlapping ends. Eachrestriction mixture contains 5 μl of NEB3 buffer from New EnglandBiolabs (NEB, Schwalbach, Germany), 20 U of EcoRI (NEB), BamHI (NEB), ina total volume of 50 μl and is incubated for 3 h at 37° C. Digestedvector DNA and PCR product are purified using the PCR purification kitfrom Qiagen.

20 ng of vector DNA and 8 ng of PCR product are ligated together with 1U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pNCOgcpE. The ligation mixture isincubated overnight at 4° C.

With 2 μl of the ligation mixture electrocompetent E. coli XL1-Bluecells are transformed.

Electrocompetent cells are prepared according to reference example 1.Electro-transformation is done according to the description in referenceexample 1.

The plasmid pNCOgcpE is transformed into electrocompetent E. coli M15(pREP4) (Zamenhof et al. J. Bacteriol. 110, 171-178 (1972)) cells asdescribed above yielding the recombinant strain M15-pNCOgcpE.

The DNA sequence of the plasmid pNCOgcpE is sequenced as described inreference example 1. The DNA sequence is found to be identical with thesequence in the database (gb AE000338).

Example 2 Construction of an Expression Vector and Production of anExpression Clone for the lytB Gene of Escherichia coli

The expression vector pQE30 (Qiagen) is isolated as described inreference example 1. The E. coli ORF lytB (accession no. gb AE000113)from basepair (bp) position 5618 to 6565 is amplified by PCR usingchromosomal E. coli DNA as template. The reaction mixture contains 25pmol of the primer 5′-TGGAGGGGATCCATGCAGATCCTGTTGGCC-3′, 25 pmol ofprimer 5′-GCATTTCTGCAGAACTTAGGC-3′, 20 ng of chromosomal DNA, 2 U of TaqDNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in atotal volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 60sec at 94° C., 60 sec at 50° C. and 60 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis. The PCRamplificate is purified with the PCR purification kit from Qiagen asdescribed in reference example 2. 1.9 μg of purified PCR product areobtained.

2.0 μg of the vector pQE30 and 1.5 μg of the purified PCR product aredigested in order to produce DNA fragments with overlapping ends. Eachrestriction mixture contains 5 μl of NEB3 buffer from NEB, 20 U of BamHI(NEB), 20 U of PstI (NEB) in a total volume of 50 μl and is incubatedfor 3 h at 37° C. Digested vector DNA and PCR product are purified usingthe PCR purification kit from Qiagen.

18 ng of vector DNA and 10 ng of PCR product are ligated together with 1U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pQElytB. The ligation mixture isincubated overnight at 4° C. 2 μl of the ligation mixture is transformedinto electrocompetent E. coli XL1-Blue cells as described in referenceexample 1. The electrocompetent cells are prepared as described inreference example 1.

The plasmid pQElytB M15 (pREP4) cells yield the recombinant strainM15-pQElytB. The DNA insert of the vector pQElytB is sequenced asdescribed in reference example 1. The DNA sequence is found to beidentical with the sequence in the database (gb AE000113).

Example 3 Preparation and Purification of the Recombinant 6× His-LytBFusion Protein of E. coli

0.5 liter of Luria Bertani (LB) medium containing 90 mg of ampicillinand 25 mg kanamycin are inoculated with 10 ml of an overnight culture ofE. coli strain M15 (pREP4) harboring plasmid pQElytB. The culture isgrown in a shaking culture at 37° C. At an optical density (600 nm) of0.7, the culture is induced with 2 mM IPTG. The culture is grown forfurther 5 h. The cells are harvested by centrifugation for 20 min at5,000 rpm and 4° C. The cells are washed with 50 mM tris hydrochloridepH 8.0, centrifuged as above and frozen at −20° C. for storage.

The cells are thawed in 20 ml of 20 mM imidazole in 100 mM trishydrochloride pH 8.0 and 0.5 M sodium chloride (standard buffer) in thepresence of 1 mg/ml lysozyme and 100 μg/ml DNasel. The mixture isincubated at 37° C. for 30 min, cooled on ice and sonified 6×10 sec witha Branson Sonifier 250 (Branson SONIC Power Company) set to 70% dutycycle output, control value of 4 output. The suspension is centrifugedat 15,000 rpm at 4° C. for 30 min. The cell free extract of recombinantLytB protein is applied on a column of Ni²⁺-Chelating sepharose FF (size2.6×6 cm, Amersham Pharmacia Biotech) previously equilibrated with 20 mMimidazole in standard buffer. The column is washed with 100 ml ofstarting buffer. LytB protein is eluted with a linear gradient of 20-500mM imidazole in 100 ml of standard buffer. LytB protein containingfractions are combined according to SDS-PAGE and dialysed overnightagainst 100 mM tris hydrochloride pH 8.0, 5 mM dithioerythritol, 0.02%sodium azide. The homogeneity of the dialysed LytB protein is judged bySDS-PAGE. The objected band at 34 kDa is in agreement with thecalculated molecular mass. 3 mg of pure enzyme were obtained.

Example 4 Construction of an Expression Vector and Production of anExpression Clone for the yjeE Gene of Escherichia coli

The expression vector pNCO113 is isolated as described in referenceexample 1.

The E. coli ORF yjeE (accession no. gb AE000489) is amplified from basepair (bp) position 3299 to 3760 by PCR using chromosomal E. coli DNA astemplate. The reaction mixture contains 25 pmol of the primer5′-GAGGAGAAATTAACCATGATGAATCGAGTAATTCC-3′, 25 pmol of the primer5′-GCGATACTGCAGCCCGCC-3′, 20 ng of chromosomal DNA, 2 U of Taq DNApolymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in atotal volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 30sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with the PCR purification kit fromQiagen as described in reference example 2.1.1 μg of purified PCRproduct are obtained.

The PCR amplificate is used as template for a second PCR reaction. Thereaction mixture contains 25 pmol of the primer5′-ACACAGAATTCATTAAAGAGGAGAAATTAACCATG-3′, 25 pmol of the primer5′-GCGATACTGCAGCCCGCC-3′, 2 μl of the first PCR amplification, 2 U ofTaq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPsin a total volume of 100 μl containing 1.5 mM MgCl₂, 50 mM KCl, 10 mMtris hydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 30sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with PCR purification kit from Qiagen asdescribed above. 1.0 μg of purified PCR product are obtained. 2.0 μg ofthe vector pNCO113 and 1.1 μg of the purified PCR product are digestedin order to produce DNA fragments with overlapping ends. Eachrestriction mixture contains 5 μl of NEB3 buffer from NEB, 20 U of EcoRI(NEB), PstI (NEB), in a total volume of 50 μl and is incubated for 3 hat 37° C. Digested vector DNA and PCR product are purified using the PCRpurification kit from Qiagen.

20 ng of vector DNA and 8 ng of PCR product are ligated together with 1U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pNCOyjeE. The ligation mixture isincubated overnight at 4° C.

2 μl of the ligation mixture is transformed into electrocompetent E.coli XL1-Blue as described in reference example 1 yielding therecombinant strain XL1-pNCOyjeE.

The DNA sequence of the plasmid pNCOyjeE is sequenced as described inreference example 1. The DNA sequence is found to be identical with thesequence in the database (gb AE000489).

Example 5 Construction of an Expression Vector and Production of anExpression Clone for the ybeB Gene of Escherichia coli

The expression vector pQE30 is isolated as described in referenceexample 1. The E. coli ORF ybeB (gb AE000168) is amplified from basepair (bp) position 6454 to 6663 by PCR using chromosomal E. coli DNA astemplate. The reaction mixture contains 25 pmol of the primer5′-CCAGGGGGGATCCATGCAGGGTAAAGC-3′, 25 pmol of the primer5′-GTTGCAGCTGCAGGCATTAACTCC-3′, 20 ng of chromosomal DNA, 2 U of Taq DNApolymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in atotal volume of 100 μl of 1.5 mM MgCl₂, 50 mM KCl, 10 mM trishydrochloride, pH 8.8 and 0.1% (w/w) Triton X-100.

The mixture is denaturated for 3 min at 95° C. Then 30 PCR cycles for 30sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. follow. Afterfurther incubation for 7 min at 72° C., the mixture is cooled to 4° C.An aliquot of 2 μl is subjected to agarose gel electrophoresis.

The PCR amplificate is purified with the PCR purification kit fromQiagen as described in reference example 2. 1.5 μg of purified PCRproduct are obtained. 2.0 μg of the vector pQE30 and 1.0 μg of thepurified PCR product are digested in order to produce DNA fragments withoverlapping ends. Each restriction mixture contains 5 μl of NEB3 bufferfrom NEB, 20 U of BamHI (NEB) and PstI (NEB), in a total volume of 50 μland is incubated for 3 h at 37° C. Digested vector DNA and PCR productare purified using the PCR purification kit from Qiagen.

20 ng of vector DNA and 6 ng of PCR product are ligated together with 1U of T4-Ligase (Gibco), 2 μl of T4-Ligase buffer (Gibco) in a totalvolume of 10 μl, yielding the plasmid pNCOyjeE. The ligation mixture isincubated overnight at 4° C.

2 μl of the ligation mixture is transformed into electrocompetent E.coli XL1-Blue as described in reference example 1 yielding therecombinant strain XL1-pQE30ybeB.

The DNA sequence of the plasmid pQEybeB is sequenced as described inreference example 1. The DNA sequence is found to be identical with thesequence in the database (gb AE000168).

Example 6 Consumption Assay of 2C-methyl-D-erythritol2,4-cyclopyrophosphate Using Protein Fraction of E. coli

Assay mixtures for direct detection of enzymatic products formed from2C-methyl-D-erythritol 2,4-cyclopyrophosphate via reversed-phaseion-pair HPLC contain 100 mM tris hydrochloride pH 8.0, 10 mM MgCl₂, 25μM of [2-¹⁴C]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate (15.8μCi/μMol) and protein in a total volume of 200 μl. They are incubated at37° C. for 4 hours. The reactions are stopped by freezing the samples inliquid nitrogen and the reaction mixtures are centrifuged. Aliquots (50μl) of the supernatant are applied to a Multospher 120 RP 18 column (5μm, crystalline silica gel, 4.6×250 mm, CS-Chromatographie Service GmbH,Langerwehe, Germany) which has been equilibrated with 10 mMtetra-n-butylammonium hydrogensulfate pH 6.0 at a flow rate of 0.75ml/min. The column is developed with 15 ml of 10 mMtetra-n-butylammonium hydrogensulfate pH 6.0 and further by a lineargradient of 0 to 42% (v/v) methanol in 45 ml of 10 mMtetra-n-butylammonium hydrogen sulfate pH 6.0. The effluent is monitoredusing a radiodetector (Beta-RAM, Biostep GmbH, Jahnsdorf, Germany). Theretention volumes of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate, anew product and isopentenyl pyrophosphate are 31.5, 40 and 59 mlrespectively. This assay can be carried out in the presence or absenceof prospective inhibitors by measuring the residual amount of startingmaterial or the amount of product and comparing the results.

Example 7 Assays with Chromoplasts of Narcissus pseudonarcissus

Chromoplasts of Narcissus pseudonarcissus are prepared according to amethod described by Kleinig and Beyer (in: Methods in Enzymology (Law,J. H. and Rilling, H. C., eds) 110, 267-273 (1985), Academic Press,London).

Assay mixtures contain 100 mM Hepes pH 7.6, 2 mM MnCl₂, 10 mM MgCl₂, 2mM NADP⁺, 20 μM FAD, 5 mM NaF, 6 mM ATP, 1 mM NADPH, 64 μM[2-¹⁴C]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate (15.8 μCi/μMol)and 1 mg chromoplasts in a total volume of 500 μl. The mixture isincubated at 30° C. for 240 min and analyzed by HPLC on the occurrenceof new metabolites by as described in example 6. Peaks of newmetabolites with retention volumes of 40, 47 and 59 ml are obtained.

Example 8 Enzymatic Preparation of a New Metabolite of theNon-Mevalonate Pathway

25 g of M15 (pREP4) cells grown overnight in M9 minimal medium (Sambrooket al., Molecular Cloning, 2nd edition (1989), Cold Spring Harbor Press)are harvested by centrifugation and resuspended in 150 ml of a solutionof 10 mM MgCl₂, 5 mM dithiothreitol and 100 mM tris hydrochloride pH8.0. 100 mg of lysozyme are added and the reaction mixture is incubatedfor 30 min at 37° C. The mixture is cooled on ice and sonified 6×10 secwith a Branson Sonifier 250 (Branson SONIC Power Company) set to 70%duty cycle output, control value of 4 output and centrifuged for 60 minat 10,000 rpm. The pellet is resuspended in 200 ml of 100 mM trishydrochloride, pH 8.0, 5 mM CoCl₂, 10 mM MgCl₂, 5 mM dithiothreitol and27.3 μM [U—¹³C₅, 2-¹⁴C]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate(0.72 μCi/μMol) are added and the mixture is incubated for 8 h at 37° C.The mixture is centrifuged at 10,000 rpm for 60 min and the supernatantis lyophylized. The dried substance is dissolved in 20 ml destinedwater. An aliquot of 100 μl is analyzed by HPLC on the occurrence of anew product peak at 40 min as described in example 6. 150 ml ethanol areadded and the mixture is centrifuged at 4,800 rpm for 30 min. The pelletis resuspended in 130 ml ethanol, 100 mg barium acetate are added to thesuspension and the suspension is kept overnight at 4° C. The mixture iscentrifuged at 4,800 rpm for 30 min and the pellet is collected bycentrifugation at 4,800 rpm for 30 min. The pellet is resuspended in 5ml destined water and beads of a cation exchanger (AG-50W-x8, sodiumform, Biorad, München) are added until a clear solution is obtained. Thesolution is passed through a filter of Whatman paper and the filtrate islyophilized. The dried substance is dissolved in 0.5 ml of D₂O.

1-31. (canceled)
 32. Optionally isotope-labelled intermediate in thenon-mevalonate isoprenoid pathway characterized in that (a) it isproducible by the reaction of 2C-methyl-D-erythritol2,4-cyclopyrophosphate with an aqueous suspension of intact or lysedplastids or bacterial cells; (b) it is separable from the aqueoussupernatant of the reaction mixture obtainable according to item (a) byreversed-phase, ion pair, high performance liquid chromatography using a4.6×250 mm C₁₈ reversed-phase column (crystalline silica gel of particlesize 5 μm, average pore size 12 nm), equilibrated with aqueous 10 mMtetra-n-butylammonium hydrogen sulfate pH 6.0 and developed with 10 mMtetra-n-butylammonium hydrogen sulfate pH 6.0 and a linear gradient of 0to 42% (v/v) methanol in 10 mM tetra-n-butyl ammonium hydrogen sulfateat pH 6.0 and ambient temperature; (c) it exhibits under the conditionsof item (b) a retention volume downstream from the retention volume of2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream from theretention volume of isopentenyl pyrophosphate; and (d) its increase inthe reaction mixture of item (a) is correlated with the decrease of2C-methyl-D-erythritol 2,4-cyclopyrophosphate as detectable in thedetection method of item (c).
 33. The intermediate according to claim 32characterized in that it is producible in a partially radiolabelled formfrom a partially ¹⁴C-labelled 2C-methyl-D-erythritol2,4-cyclopyrophosphate.
 34. The intermediate according to claim 32characterized in that it is producible as a [U—¹³C]-compound from[U—¹³C₅]-2C-methyl-D-erythritol 2,4-cyclopyrophosphate.
 35. Theintermediate according to claim 32 characterized by a retention volumeof 1.27±0.15 times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphateand 0.68±0.06 times that of isopentenyl pyrophosphate or of 1.50±0.16times that of 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and0.80±0.06 times that of isopentenyl pyrophosphate under the conditionsdescribed in claim 1 item b.
 36. The intermediate according to claim 35characterized by a retention volume of about 1.27 times that of2C-methyl-D-erythritol 2,4-cyclopyrophosphate and about 0.68 times thatof isopentenyl pyrophosphate or of about 1.50 times that of2C-methyl-D-erythritol 2,4-cyclopyrophosphate and about 0.80 times thatof isopentenyl pyrophosphate under the conditions described in claim 1item b.
 37. A process for producing an intermediate in thenon-mevalonate isoprenoid pathway by (a) adding 2C-methyl-D-erythritol2,4-cyclopyrophosphate to an aqueous suspension of intact or lysedplastids or bacterial cells or of a protein fraction thereof; (b)incubating the obtained mixture for a predetermined period of time at apredetermined temperature; (c) optionally disrupting the plastids orcells; (d) separating insoluble components and polymer components fromthe mixture; (e) subjecting the obtained aqueous solution, optionallyafter concentration, to a reversed phase ion pair high performanceliquid chromatography; (f) separating a fraction containing a metabolitehaving a retention volume downstream from the retention volume of2C-methyl-D-erythritol 2,4-cyclopyrophosphate and upstream from theretention volume of isopentenyl pyrophosphate; and (g) isolating theintermediate from said fraction.
 38. The process according to claim 37,wherein a radioactively labelled starting material is used and thedetection in step (f) is carried out with a radio detector.
 39. Theprocess according to claim 37 characterized in that a cobalt (II) saltis added in step (a).
 40. Use of the intermediate according to claim 32for screening for enzymes of the non-mevalonate pathway (a) eitherbetween 2C-methyl-D-erythritol 2,4-cyclopyrophosphate and saidintermediate; or (b) between said intermediate and isopentenylpyrophosphate or dimethylallyl pyrophosphate.
 41. An enzyme in a formthat is functional in the biosynthetic conversion of2C-methyl-D-erythritol 2,4-cyclopyrophosphate into the intermediateaccording to claim 32 within the non-mevalonate isoprenoid pathwayselected from the group of enzymes coded by (a) gcpE of E. coli or bygenes orthologous thereto; (b) lytB of E. coli or by genes orthologousthereto; (c) yjeE of E. coli or by genes orthologous thereto; (d) ybeBof E. coli or by genes orthologous thereto.
 42. An isolated, purifiednucleic acid coding for an enzyme according to claim
 41. 43. A DNAvector containing the sequence of the nucleic acid according to claim42.
 44. Recombinant cell containing the vector according to claim 43,wherein said cell is selected from the group consisting of bacterial,protozoal, fungal, plant, insect and mammalian cells.
 45. A seedcomprising a plant cell as defined in claim 44.